Cysteine protease inhibitors

ABSTRACT

Compounds of the formula I 
     
       
         
         
             
             
         
       
     
     wherein
     R 2a  and R 2b  are independently H, halo, C 1 -C 4 alkyl, C 1 -C 4 haloalkyl or C 1 -C 4 alkoxy, or R 2a  and R 2b  together with the carbon atom to which they are attached form a C 3 -C 6 cycloalkyl;   R 3  is a C 5 -C 10  alkyl, optionally substituted with 1-3 substituents independently selected from halo, C 1 -C 4 haloalkyl, C 1 -C 4 alkoxy, C 1 -C 4 haloalkoxy; or   R 3  is a C 2 -C 4 alkyl chain with at least 2 chloro or 3 fluoro substituents; or   R 3  is C 3 -C 7 cycloalkylmethyl, optionally substituted with 1-3 substituents independently selected from C 1 -C 4 alkyl, halo, C 1 -C 4 haloalkyl, C 1 -C 4 alkoxy, C 1 -C 4 haloalkoxy;   R 4  is C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, C 1 -C 6 alkylamino, C 1 -C 6 dialkylamino or;   R 4  is Het or Carbocyclyl, either of which is optionally substituted with 1-3 substituents R 4  is Het, carbocyclyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy or C 1 -C 6 haloalkoxy;   n is 1, 2 or 3;   for the use in the prophylaxis or treatment of a disorder characterised by inappropriate expression or activation of cathepsin S.

This application is a Continuation of co-pending application Ser. No.13/704,441 filed on Dec. 14, 2012, and for which priority is claimedunder 35 U.S.C. §120. Application Ser. No. 13/704,441 is the NationalPhase of PCT International Application No. PCT/IB2011/052613 filed onJun. 16, 2011 under 35 U.S.C. §371. This application also claimspriority of Application No. 1050618-6 filed in Sweden on Jun. 16, 2010under 35 U.S.C. §119. The entire contents of each of theabove-identified applications are hereby incorporated by reference intheir entirety.

TECHNICAL FIELD

This invention relates to inhibitors of cathepsin S, and their use inthe treatment of disorders involving cathepsin S such as autoimmunedisorders, allergy and chronic pain conditions.

BACKGROUND TO THE INVENTION

The papain superfamily of cysteine proteases are widely distributed indiverse species including mammals, invertebrates, protozoa, plants andbacteria. A number of mammalian cathepsin enzymes, including cathepsinsB, F, H, K, L, O, S, and W, have been ascribed to this superfamily, andinappropriate regulation of their activity has been implicated in anumber of metabolic disorders including arthritis, muscular dystrophy,inflammation, glomerulonephritis and tumour invasion. Pathogeniccathepsin like enzymes include the bacterial gingipains, the malarialfalcipains I, II, III et seq and cysteine proteases from Pneumocystiscarinii, Trypanosoma cruzei and brucei, Crithidia fusiculata,Schistosoma spp.

In WO 97/40066, the use of inhibitors against Cathepsin S is described.The inhibition of this enzyme is suggested to prevent or treat diseasecaused by protease activity. Cathepsin S is a highly active cysteineprotease belonging to the papain superfamily. Its primary structure is57%, 41% and 45% homologous with human cathepsin L and H and the plantcysteine protease papain respectively, although only 31% homologous withcathepsin B. It is found mainly in B cells, dendritic cells andmacrophages and this limited occurrence suggests the potentialinvolvement of this enzyme in the pathogenesis of degenerative disease.Moreover, it has been found that destruction of Ii by proteolysis isrequired for MHC class II molecules to bind antigenic peptides, and fortransport of the resulting complex to the cell surface. Furthermore, ithas been found that Cathepsin S is essential in B cells for effective Iiproteolysis necessary to render class II molecules competent for bindingpeptides. Therefore, the inhibition of this enzyme may be useful inmodulating class II-restricted immune response (WO 97/40066). Otherdisorders in which cathepsin S is implicated are asthma, chronicobstructive pulmonary disease, endometriosis and chronic pain.

BRIEF DESCRIPTION OF THE INVENTION

According to a first aspect of the invention there is provided acompound of the formula I:

-   -   wherein    -   R^(2a) and R^(2b) are independently selected from H, halo,        C₁-C₄alkyl, C₁-C₄haloalkyl, C₁-C₄alkoxy, or R^(2a) and R^(2b)        together with the carbon atom to which they are attached form a        C₃-C₆cycloalkyl;    -   R³ is a C₅-C₁₀ alkyl, optionally substituted with 1-3        substituents independently selected from halo, C₁-C₄haloalkyl,        C₁-C₄alkoxy, C₁-C₄haloalkoxy; or    -   R³ is a C₂-C₄alkyl chain with at least 2 chloro or 3 fluoro        substituents; or    -   R³ is C₃-C₇cycloalkylmethyl, optionally substituted with 1-3        substituents independently selected from C₁-C₄alkyl, halo,        C₁-C₄haloalkyl, C₁-C₄alkoxy, C₁-C₄haloalkoxy;    -   R⁴ is C₁-C₆alkyl, C₁-C₆haloalkyl, C₁-C₆alkoxy, C₁-C₆haloalkoxy,        C₁-C₆alkylamino, C₁-C₆dialkylamino or;    -   R⁴ is Het or Carbocyclyl, either of which is optionally        substituted with 1-3 substituents independently selected from:        -   halo, azido, cyano, hydroxy, oxo, C₁-C₄alkyl,            C₁-C₄haloalkyl, C₁-C₄alkoxy, C₁-C₄haloalkoxy,            C₁-C₄alkoxycarbonyl, C₁-C₄alkylcarbonyl, C₁-C₄alkylsulfonyl,            C₁-C₄alkylsulfonylamino, aminosulphonyl, NRaRb, C(═O)NRaRb,            NRaC(═O)Rb, NRaC(═O)ORb, NRa(C═O)NRaRb, wherein oxo as            substituent may be present only where valence so permits;        -   and/or wherein in the Het or Carbocyclyl group is optionally            substituted with a group of the formula —X—R⁵; wherein            -   X is C₁-C₄alkylene or a 1-4 membered linkage comprising                0-3 methylene groups disposed adjacent to, or on either                side of a —CH(CH₃)—, —C(CH₃)₂—, CF₂—, —O—, —CH═CH—,                —C≡C—, —NRa—, —C(═O)NRa—, —NRaC(═O)—, —C(═O)NRa—,                —NRaS(═O)₂—, —S(═O)₂NRa—, ester, urea or carbamate                function;            -   R⁵ is H, C₁-C₄alkyl or a monocyclic ring selected from                C₃-C₆cycloalkyl, C₃-C₆cycloalkenyl, phenyl,                pyrrolidinyl, piperidinyl, morpholinyl, thiomorpholinyl,                piperazinyl, indolinyl, pyranyl, tetrahydropyranyl,                tetrahydrothiopyranyl, thiopyranyl, furanyl,                tetrahydrofuranyl, thienyl, pyrrolyl, oxazolyl,                isoxazolyl, thiazolyl, imidazolyl, pyridinyl,                pyrimidinyl, pyrazinyl, pyridazinyl, tetrazolyl,                pyrazolyl, indolyl, the C₁-C₄alkyl or monocyclic ring                being optionally substituted with one to three                substituents selected from:                -   halo, azido, cyano, hydroxy, C₁-C₄alkyl,                    C₁-C₄haloalkyl, C₁-C₄alkoxy, C₁-C₄haloalkoxy,                    C₁-C₄alkoxycarbonyl, C₁-C₄alkylcarbonyl, amino,                    C₁-C₄alkylamino, C₁-C₄dialkylamino,                    C₁-C₄alkylsulfonyl, C₁-C₄alkylsulfonylamino,                    aminocarbonyl, aminosulphonyl;    -   n is 1, 2 or 3;    -   Ra and Rb are independently H, C₁-C₄alkyl;    -   Het is a stable, monocyclic or bicyclic, saturated, partially        saturated or aromatic ring system containing 1-4 heteroatoms        independently selected from O, S and N, each ring having 5 or 6        ring atoms;    -   Carbocyclyl is C₃-C₆cycloalkyl, C₅-C₆cycloalkenyl or phenyl;        or a pharmaceutically acceptable salt, hydrate or N-oxide        thereof.

Typical embodiments of the invention include compounds wherein n is 1 or2, i.e. compounds having a 1,1-cyclopropylenyl or 1,1-cyclobutylenylring, thus providing compounds according to formula Ia or Ibrespectively.

The substituents R^(2a) and R^(2b) may be attached to any of the atomsof the 1,1-cycloalkylenyl group.

In some embodiments of the invention the 1,1-cycloalkylenyl ring isunsubstituted, i.e. R^(2a) and R^(2b) are both H.

In some embodiments of the invention where n is 2, the1,1-cyclobutylenyl ring is substituted with one or two substituents,i.e. one or both of R^(2a) and R^(2b) is/are not H. Typically accordingto these embodiments, the substituent(s) is/are located in the2-position of the cyclobutylenyl ring, thus providing compoundsaccording to formula Ic:

In some embodiments where n is 2, R^(2a) and R^(2b) are both hydrogen.In alternative embodiments where n is 2, one of R^(2a) and R^(2b) is Hand the other is halo, C₁-C₄alkyl, C₁-C₄haloalkyl or C₁-C₄alkoxy. Infurther alternative embodiments where n is 2, R^(2a) and R^(2b) are bothhalo, C₁-C₄alkyl or C₁-C₄haloalkyl.

Typical embodiments where n is 2, include compounds wherein one ofR^(2a) and R^(2b) is H, and the other is Cl, F, CF₃ or MeO.

In a preferred embodiment where n is 2, one of R^(2a) and R^(2b) is H,and the other is F. Specially preferred according to this embodiment arecompounds having the stereochemistry shown in formula Id:

In some embodiments where n is 2, R^(2a) and R^(2b) are both F.Compounds of this aspect have the formula Ie:

In other embodiments where n is 2, one of R^(2a) and R^(2b) is H, andthe other is Cl, F, CF₃ or MeO.

In some embodiments R^(2a) and R^(2b) together with the carbon atom towhich they are attached form a C₃-C₆cycloalkyl;

In some embodiments R³ is cycloalkylalkyl, optionally substituted, forexample with halo, (such as F) or alkoxy (such as MeO). Exemplaryspecies include 1-methylcyclopentylmethyl, 1-methylcyclohexylmethyl,1-methylcyclobutylmethyl, 1-methyl-3,3-difluorocyclobutylmethyl,1-methyl-4,4-difluorocyclohexylmethyl, cyclopropylmethyl or1-methyl-3,3-difluorocyclopentylmethyl.

Representative R³ species include t-butylmethyl, cyclobutylmethyl,1-methylcyclobutylmethyl and 1-methylcyclopentylmethyl, any of which isoptionally substituted with one or two F or MeO. Representative speciesare 1-fluorocyclobutylmethyl and 1-fluorocyclopentylmethyl.

Further representative R³ species include 1-methylcyclopentylmethyl and1-fluorocyclopentylmethyl,

Other embodiments have R³ as a straight or branched alkyl chain of 5-10C-atoms, optionally substituted with 1-3 halo, (e.g. Cl or F), or aC₁-C₄alkoxy (e.g. MeO). Exemplary species include 2,2-dimethylpropyl,3,3-dimethylpentyl, 2,2,3,3-tetramethylbutyl. Exemplary species ofhalogenated alkyl include 2,2-dichioroethyl, 3,3,3-trifluoropropyl,2,2-trifluoromethylethyl, or 2,2,2-trifluoroethyl.

One embodiment of the invention include compounds wherein R⁴ isC₁-C₆alkyl, such as ethyl, isopropyl or tert.butyl.

Another embodiment includes compounds wherein R⁴ is C₁-C₆haloalkyl, suchas chloroalkyl or fluoroalkyl. Typically according to this embodiment,R⁴ is mono, di or tri fluoroC₁-C₆alkyl, such as monofluoropropyl,difluoropropyl, trifluoropropyl or trifluorobutyl.

Another embodiment of the invention includes compounds wherein R⁴ isC₁-C₆alkoxy or C₁-C₆haloalkoxy, thus affording compounds having acarbamate function as shown in formula II,

wherein R^(4′) is C₁-C₆alkyl or C₁-C₆haloalkyl. Typically according tothis embodiment, R^(4′) is methyl or ethyl.

One embodiment of the invention includes of compounds of formula II,wherein n is 2 and at least one of R^(2a) and R^(2b) is halo,C₁-C₄alkyl, C₁-C₄haloalkyl or C₁-C₄alkoxy. Typically according to thisembodiment, one of R^(2a) and R^(2b) is H, and the other is chloro,fluoro, trifluoromethyl or methoxy; especially fluoro or methoxy.Preferably according to this embodiment, R^(2a) and R^(2b) are locatedin the 2-position of the cyclobutylenyl group.

Preferred embodiments of the invention include compounds of formula IIwherein n is 2, one of R^(2a) and R^(2b) is H, and the other is F.Specially preferred according to this embodiment are compounds whereinthe F-atom is located in the 2-position of the cyclobutylenyl group, andhaving the stereochemistry shown in formula IIa:

Other embodiments of the invention include compounds of formula II,wherein n is 2, R^(2a) and R^(2b) are both F, thus providing compoundsof formula IIb:

In further embodiments of compounds of formula II, R^(2a) and R^(2b) areboth H, thus providing compounds of formula IIc:

Preferred compounds according to this embodiment are those wherein n is2, i.e. a cyclobutylenyl group is formed, thus providing compound of theformula IId:

In some embodiments, R⁴ is morpholinyl, piperidinyl, piperazinyl,cyclopentyl, cyclohexyl or pyridinyl, any of which is optionallysubstituted with halo, hydroxy, C₁-C₄alkyl, C₁-C₄haloalkyl, C₁-C₄alkoxy,C₁-C₄haloalkoxy, amino, C₁-C₄alkylamino, di(C₁-C₄-alkyl)amino orNRaS(═O)_(m)Rq;

-   -   where Ra is H or C₁-C₄alkyl;    -   Rq is C₁-C₄alkyl, Het or Carbocyclyl, any of which is optionally        substituted with C₁-C₄alkyl, halo, C₁-C₄haloalkyl, C₁-C₄ alkoxy;        and    -   m is 0, 1 or 2.

In other embodiments R⁴ is an optionally substituted thiazolyl, such asthiazol-5-yl, optionally substituted with C₁-C₄ alkyl (e.g. Me) halo(e.g. F) or C₁-C₄alkoxy (e.g. MeO).

In other embodiments R⁴ is morpholin-4-yl.

In some embodiments, R⁴ is linked to the adjacent backbone amide througha ring nitrogen, thereby defining a urea function. A representativespecies is morpholin-4-yl. A further representative species has thepartial structure:

where X is C and Rt is hydroxy, fluoro, C₁-C₄alkyl, e.g. gem-methyl,C₁-C₄alkoxy (e.g. MeO) C₁-C₄haloalkyl (e.g. CF₃) or an NRk-S(═O)₂Rsfunction where Rs is C₁-C₄ alkyl, Het or Carbocyclyl, any of which isoptionally substituted with 1-3 C₁-C₄alkyl (e.g. Me), halo (e.g. F),C₁-C₄haloalkyl (e.g. CF₃), or C₁-C₄ alkoxy (e.g. MeO). Alternatively Xis N and Rt is C₁-C₄alkyl (e.g. Me) or an —NRk-S(═O)₂Rs function whereRs is C₁-C₄alkyl, Het or Carbocyclyl, any of which is optionallysubstituted with 1-3 C₁-C₄alkyl (e.g. Me), halo (e.g. F), C₁-C₄haloalkyl(e.g. CF₃), or C₁-C₄alkoxy (e.g. MeO).

In some embodiments R⁴ is piperazin-1-yl or piperidin-1-yl, either ofwhich is substituted at the 4 position or piperidin-4-yl substituted atthe 1 position; in each case the substituent is selected fromNHS(═O)₂Carbocyclyl or NHS(═O)₂Het, wherein the Carbocyclyl or Het isoptionally substituted with halo, C₁-C₄alkyl, C₁-C₄haloalkyl orC₁-C₄alkyoxy.

In other embodiments, R⁴ is cyclohexyl or piperazin-1-yl substituted atthe 4 position with halo, amino, C₁-C₄alkylamino di-(C₁-C₄alkyl)amino orhydroxy.

In some embodiments R⁴ is substituted phenyl.

In some embodiments R⁴ is phenyl which is substituted with 1, two orthree substituents independently selected from halo, hydroxy,C₁-C₄alkyl, C₁-C₄haloalkyl, cyano, C₁-C₄alkylC(═O)NH— and C₁-C₄alkoxy.

Representative species include phenyl substituted with m-fluoro,p-fluoro, p-hydroxy, p-hydroxy-m-chloro, p-hydroxy-m-fluoro,p-hydroxy-m-methoxy, p-hydroxy-m-methyl, bis-p-chloro-p-hydroxy,m-cyano, p-acetamido or o-fluoro-p-hydroxy.

As defined above, R⁴ may be substituted with a group of the formulaX—R⁵, where R⁵ is H, optionally substituted C₁-C₄alkyl or an optionallysubstituted monocyclic ring which is spaced from the R⁴ ring by thedivalent X linker.

Linker X may comprise a straight chain C₁-C₄alkylene, such as ethyleneor methylene. Alternatively, the linker may comprise a divalent functionselected from CH(CH₃), C(CH₃)₂, CF₂, ethene, ethyne, C₀-C₄alkylamine,C₀-C₄alkylamide, sulphonamide, ester, ether, urea or carbamate, whichmay be bonded direct to R⁴ and/or R⁵ or may have one or more methylenegroups between the function and R⁴ and/or between the function and R⁵.The total length of the linker (including any methylene groups betweenthe function and R⁴ and/or R⁵ is 1-4 chain atoms, preferably one tothree.

Divalent functions in the X-linker containing multiple hetero atoms,such as amide, sulphonamide, ester or carbamate may be disposed ineither orientation, for example —O(C═O)NH— or —NH(═O)O— in the case of acarbamate. The expressions C₀-C₄alkylamine and C₀-C₄alkylamide in thecontext of the X-linker mean that an alkyl group (or H in the case ofC₀) branches from the nitrogen atom, for example —NH—, —N(CH₃)—,—NHC(═O)—, —N(CH₃)C(═O), —C(═O)N(CH₃)— etc.

Still further R⁴ groups include those described in WO0664206 thecontents of which are incorporated by reference. Notable R⁴ groups fromthis reference include those with the partial structures:

whereinR^(4″) is H, halo, OC₁-C₄alkyl, C(═O)NRkR1, NRkC(═O)C₁-C₄alkyl,NRkC(═O)NRkR1 or —NRkC(═O)OC₁-C₄alkyl, or NHC(═O)OMe,Rk and Rl are independently H, C₁-C₄alkyl or C(═O)C₁-C₄alkyl or Rk, Rland an adjacent N atom to which they are both attached defines a cyclicamine selected from pyrrolidinyl, piperidinyl, morpholinyl, piperazinylor N-methylpiperazinyl.

Favoured subsets include those wherein R^(4″) is fluoro, methoxy,dimethylcarbamoyl, NHC(═O)Me, —NHC(═O)NHCH₃, NHC(═O)N(CH₃)₂, NHC(═O)OMeor a cyclic amine.

Other R⁴ embodiments within WO0664206 have the partial structures:

where Rm is —NRaSO_(m)R⁵ or —NHC(═O)NRkR1;Rk, Rl and the N atom to which they are both attached defines a cyclicamine selected from pyrrolidinyl, piperidinyl, morpholinyl, piperazinylor N-methylpiperazinyl;and R^(4″) is H, C₁-C₄alkyl, C₁-C₄haloalkyl, halo, cyano, hydroxyl orC₁-C₄alkoxy.

Favoured subsets include those wherein R⁵ is C₁-C₄alkyl, such as methyl,ethyl or i-propyl or t-butyl; halogenated C₁-C₄alkyl such astrifluoromethyl; C₃-C₆cycloalkyl, such as cyclopropyl or cyclohexyl; orphenyl or benzyl, any of which is optionally substituted withC₁-C₄alkyl, C₁-C₄haloalkyl, halo, cyano, C₁-C₄alkoxy.

Other R⁴ embodiments within WO0664206 have the partial structures:

where Rx is independently selected from Me, F, Cl, CF₃ and OMe, and n isindependently 0, 1 or 2.

A favoured subset has the partial structure:

Ra is H or methyl,

Rp is H, Me, F,

Rx is independently selected from Me, F, Cl, CF₃ and OMe, and n is 0, 1or 2;especially with the partial structure:

where Rx is H, F, Me.

Still further R⁴ groups described in WO06/64206 include:

where Rx is H, F, Cl, CF₃, Me, OMe, Rz is CH, NH, NMe or O and the Satom is optionally oxidised to

or preferably

Still further R⁴ groups described in WO06/64206 include:

where Ry is H, C₁-C₄alkyl, amino, NHC₁-C₄alkyl (such as methylamino),N(C₁-C₄alkyl)₂ such as dimethylamino), NHC(═O)C₁-C₄alkyl (such asacetamido);ring nitrogens are optionally substituted with C₁-C₄alkyl (such asmethyl, ethyl or t-butyl), or C(═O)C₁-C₄alkyl (such as acetyl); and

Rx is H, F, Cl, CF₃, Me, OMe.

Still further R⁴ groups described in WO06/64206 include:

where Rx is independently H, F, Cl CF₃ or OMe;one or both ring nitrogens are optionally substituted with C₁-C₄alkyl(such as methyl, ethyl or t-butyl), or C(═O)C₁-C₄ alkyl (such asacetyl);O′ is absent (i.e. 2 hydrogen atoms) or O.

Further typical R⁴ group include:

wherein Het* is a 5 or 6-membered, saturated, partially unsaturated oraromatic heterocycle containing 1-3 heteroatoms independently selectedfrom S, O and N.

Preferred compounds of the invention are those having the formula If:

wherein R^(3′) is CH₃ or F, R^(2a) and R^(2b) are independently H or F,and R⁴ is C₁-C₆alkyl, C₁-C₆haloalkyl or C₃-C₆cycloalkyl, whereinC₃-C₆cycloalkyl is optionally substituted with methyl or CF₃.

Further preferred compounds of the invention are those having theformula IIe:

wherein R^(3′) is CH₃ or F, R^(2a) and R^(2b) are independently H or F,and R^(4′) is C₁-C₆alkyl, C₁-C₆haloalkyl.

The compounds of formula I are characterised by various advantageouspharmaceutical properties and exhibit at least one improved property inview of the compounds of the prior art. In particular, the inhibitors ofthe present invention are superior in one or more of the followingpharmacological related properties, i.e. potency, decreasedcytotoxicity, improved pharmacokinetics, acceptable dosage and pillburden.

Without in any way wishing to be bound by theory, or the ascription oftentative binding modes for specific variables, P1, P2 and P3 as usedherein are provided for convenience only and have their conventionalmeanings and denote those portions of the inhibitor believed to fill theS1, S2 and S3 subsites respectively of the enzyme, where S1 is adjacentthe cleavage site and S3 remote from the cleavage site.

A further aspect of the invention comprises a method employing thecompounds of formula I for the prophylaxis or treatment of diseasescaused by aberrant expression or activation of cathepsin, i.e. diseasesor conditions alleviated or modified by inhibition of cathepsin S,preferably without substantial concomitant inhibition of other membersof the papain superfamily.

In a preferred aspect, the invention concerns a method employing thecompounds of formula I or any subgroup thereof as specified herein, forthe treatment of diseases caused by aberrant expression or activation ofcathepsin, i.e. diseases or conditions alleviated or modified byinhibition of cathepsin S, preferably without substantial concomitantinhibition of other members of the papain superfamily.

A further aspect of the invention provides the use of the compounds offormula I prophylaxis or treatment of diseases caused by aberrantexpression or activation of cathepsin, ie diseases or conditionsalleviated or modified by inhibition of cathepsin S, preferably withoutsubstantial concomitant inhibition of other members of the papainsuperfamily.

In a preferred aspect, the invention concerns the use of compounds offormula I or any subgroup thereof as specified herein, for the treatmentof diseases caused by aberrant expression or activation of cathepsin,i.e. diseases or conditions alleviated or modified by inhibition ofcathepsin S, preferably without substantial concomitant inhibition ofother members of the papain superfamily

A further aspect of the invention provides the use of the compounds offormula I for the manufacture of a medicament for the prophylaxis ortreatment of diseases caused by aberrant expression or activation ofcathepsin S, i.e. diseases or conditions alleviated or modified byinhibition of cathepsin S, preferably without substantial concomitantinhibition of other members of the papain superfamily.

In a preferred aspect, the invention concerns the use of compounds offormula I or any subgroup thereof as specified herein, for themanufacture of a medicament for the treatment of diseases caused byaberrant expression or activation of cathepsin S, i.e. diseases orconditions alleviated or modified by inhibition of cathepsin S,preferably without substantial concomitant inhibition of other membersof the papain superfamily.

Examples of such diseases or conditions defined in the immediatelypreceding three paragraphs include those enumerated in WO 97/40066, suchas autoimmune diseases, allergies, such as asthma and hay fever,multiple sclerosis, rheumatoid arthritis and the like. A further exampleis the treatment of endometriasis, and especially chronic pain, asdisclosed in WO03/20287. The invention further provides the use of thecompounds of formula I or any subgroup of formula I in therapy and inthe manufacture of a medicament for the treatment of diseases orconditions alleviated or moderated by inhibition of cathepsin S.

In one series of embodiments, the methods are employed to treat mammals,particularly humans at risk of, or afflicted with, autoimmune disease.By autoimmunity is meant the phenomenon in which the host's immuneresponse is turned against its own constituent parts, resulting inpathology. Many human autoimmune diseases are associated with certainclass II MHC-complexes. This association occurs because the structuresrecognized by T cells, the cells that cause autoimmunity, are complexescomprised of class II MHC molecules and antigenic peptides. Autoimmunedisease can result when T cells react with the host's class II MHCmolecules when complexed with peptides derived from the host's own geneproducts. If these class II MHC/antigenic peptide complexes areinhibited from being formed, the autoimmune response is reduced orsuppressed. Any autoimmune disease in which class II MHC/antigeniccomplexes play a role may be treated according to the methods of thepresent invention.

Such autoimmune diseases include, e.g., juvenile onset diabetes (insulindependent), multiple sclerosis, pemphigus vulgaris, Graves' disease,myasthenia gravis, systemic lupus erythematosus, rheumatoid arthritisand Hashimoto's thyroiditis.

In another series of embodiments, the methods are employed to treatmammals, particularly humans, at risk of, or afflicted with, allergicresponses. By “allergic response” is meant the phenomenon in which thehost's immune response to a particular antigen is unnecessary ordisproportionate, resulting in pathology. Allergies are well known inthe art, and the term “allergic response” is used herein in accordancewith standard usage in the medical field.

Examples of allergies include, but are not limited to, allergies topollen, “ragweed,” shellfish, domestic animals (e.g., cats and dogs),bee venom, house dust mite allergens and the like. Another particularlycontemplated allergic response is that which causes asthma. Allergicresponses may occur, in man, because T cells recognize particular classII MHC/antigenic peptide complexes. If these class II MHC/antigenicpeptide complexes are inhibited from being formed, the allergic responseis reduced or suppressed. Any allergic response in which class IIMHC/antigenic peptide complexes play a role may be treated according tothe methods of the present invention. Immunosuppression by the methodsof the present invention will typically be a prophylactic or therapeutictreatment for severe or life-threatening allergic responses, as mayarise during asthmatic attacks or anaphylactic shock. Preferably, thetreatment is a therapeutic treatment.

In another series of embodiments, the methods are employed to treatmammals, particularly humans, which have undergone, or are about toundergo, an organ transplant or tissue graft. In tissue transplantation(e.g., kidney, lung, liver, heart) or skin grafting, when there is amismatch between the class II MHC genotypes (HLA types) of the donor andrecipient, there may be a severe “allogeneic” immune response againstthe donor tissues which results from the presence of non-self orallogeneic class II MHC molecules presenting antigenic peptides on thesurface of donor cells. To the extent that this response is dependentupon the formation of class II MHC/antigenic peptide complexes,inhibition of cathepsin S may suppress this response and mitigate thetissue rejection. An inhibitor of cathepsin S can be used alone or inconjunction with other therapeutic agents, e.g., as an adjunct tocyclosporin A and/or antilymphocyte gamma globulin, to achieveimmunosuppression and promote graft survival. Preferably, administrationis accomplished by systemic application to the host before and/or aftersurgery. Alternatively or in addition, perfusion of the donor organ ortissue, either prior or subsequent to transplantation or grafting, maybe effective.

The above embodiments have been illustrated with an MHC class IImechanism but the invention is not limited to this mechanism of action.Suppression of cathepsin S as a treatment of COPD or chronic pain maynot, for example, involve MHC class II at all.

A related aspect of the invention is directed to a method of treating apatient undergoing a therapy wherein the therapy causes an immuneresponse, preferably a deleterious immune response, in the patientcomprising administering to the patient a compound of Formula I or apharmaceutically acceptable salt, n-oxide or hydrate thereof. Typically,the immune response is mediated by MHC class II molecules. The compoundof this invention can be administered prior to, simultaneously, or afterthe therapy. Typically, the therapy involves treatment with a biologic,such as a protein, preferably an antibody, more preferably a monoclonalantibody. More preferrably, the biologic is Remicade®, Refacto®,ReferonA®, Factor VIII, Factor VII, Betaseron®, Epogen®, Enbrel®,Interferon beta, Botox®, Fabrazyme®, Elspar®, Cerezyme®, Myobloc®,Aldurazyrne®, Verluma®, Interferon alpha, Humira®, Aranesp®, Zevalin® orOKT3. Alternatively the treatment involves use of heparin, low molecularweight heparin, procainamide or hydralazine.

Assays for the assessment of cathepsin S inhibitors in the treatment ofchronic pain, including neuropathic or inflammatory pain are asdescribed in WO 03/20287.

Currently preferred indications treatable in accordance with the presentinvention include:

Psoriasis;

Autoimmune indications, including idiopathic thrombocytopenic purpura(ITP), rheumatoid arthritis (RA), multiple sclerosis (MS), myastheniagravis (MG), Sjögrens syndrome, Grave's disease and systemic lupuserythematosis (SLE);Non-autoimmune indications include allergic rhinitis, asthma,artherosclerosis, chronic obstructive pulmonary disease (COPD) andchronic pain.

The compounds of the invention can form salts which form an additionalaspect of the invention. Appropriate pharmaceutically acceptable saltsof the compounds of the invention include salts of organic acids,especially carboxylic acids, including but not limited to acetate,trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate,malate, pantothenate, isethionate, adipate, alginate, aspartate,benzoate, butyrate, digluconate, cyclopentanate, glucoheptanate,glycerophosphate, oxalate, heptanoate, hexanoate, fumarate, nicotinate,palmoate, pectinate, 3-phenylpropionate, picrate, pivalate, propionate,tartrate, lactobionate, pivolate, camphorate, undecanoate and succinate,organic sulphonic acids such as methanesulphonate, ethanesulphonate,2-hydroxyethane sulphonate, camphorsulphonate, 2-naphthalenesulphonate,benzenesulphonate, p-chlorobenzenesulphonate and p-toluenesulphonate;and inorganic acids such as hydrochloride, hydrobromide, hydroiodide,sulphate, bisulphate, hemisulphate, thiocyanate, persulphate, phosphoricand sulphonic acids.

The compounds of the invention may in some cases be isolated as thehydrate. Hydrates are typically prepared by recrystallisation from anaqueous/organic solvent mixture using organic solvents such as dioxin,tetrahydrofuran or methanol. Hydrates can also be generated in situ byadministration of the corresponding keton to a patient.

The N-oxides of compounds of the invention can be prepared by methodsknown to those of ordinary skill in the art. For example, N-oxides canbe prepared by treating an unoxidized form of the compound of theinvention with an oxidizing agent (e.g., trifluoroperacetic acid,permaleic acid, perbenzoic acid, peracetic acid,meta-chloroperoxybenzoic acid, or the like) in a suitable inert organicsolvent (e.g., a halogenated hydrocarbon such as dichloromethane) atapproximately 0° C. Alternatively, the N-oxides of the compounds of theinvention can be prepared from the N-oxide of an appropriate startingmaterial.

Compounds of the invention in unoxidized form can be prepared fromN-oxides of the corresponding compounds of the invention by treatingwith a reducing agent (e.g., sulphur, sulphur dioxide, triphenylphosphine, lithium borohydride, sodium borohydride, phosphorusdichloride, tribromide, or the like) in an suitable inert organicsolvent (e.g., acetonitrile, ethanol, aqueous dioxane, or the like) at 0to 80° C.

The present invention also includes isotope-labelled compounds offormula I or any subgroup of formula I, wherein one or more of the atomsis replaced by an isotope of that atom, i.e. an atom having the sameatomic number as, but an atomic mass different from, the one(s)typically found in nature. Examples of isotopes that may be incorporatedinto the compounds of formula I or any subgroup of formula I, includebut are not limited to isotopes of hydrogen, such as ²H and ³H (alsodenoted D for deuterium and T for tritium respectively), carbon, such as¹¹C, ¹³C and ¹⁴C, nitrogen, such as ¹³N and ¹⁵N, oxygen, such as ¹⁵O,¹⁷O and ¹⁸O, phosphorus, such as ³¹P and ³²P, sulphur, such as ³⁵S,fluorine, such as ¹⁸F, chlorine, such as ³⁶Cl, bromine such as ⁷⁵Br,⁷⁶Br, ⁷⁷Br and ⁸²Br, and iodine, such as ¹²³I, ¹²⁴I, ¹²⁵I and ¹³¹I.

The choice of isotope included in an isotope-labelled compound willdepend on the specific application of that compound. For example, fordrug or substrate tissue distribution assays, compounds wherein aradioactive isotope such as ³H or ¹⁴C is incorporated will generally bemost useful. For radio-imaging applications, for example positronemission tomography (PET) a positron emitting isotope such as ¹¹C, ¹⁸F,¹³N or ¹⁵O will be useful. The incorporation of a heavier isotope, suchas deuterium, i.e. ²H, may provide greater metabolic stability to acompound of formula I or any subgroup of formula I, which may result in,for example, an increased in vivo half life of the compound or reduceddosage requirements. For example, ²H isotope(s) are typicallyincorporated at position(s) disposed to metabolic liability. In thecompounds of the present invention, suitable positions for incorporationof ²H isotopes are e.g. as substituents to the 1,1-cycloalkylene group,i.e. one or both of R^(2a) and R^(2b) is ²H.

Isotopically labelled compounds of formula I or any subgroup of formulaI can be prepared by processes analogous to those described in theSchemes and/or Examples herein below by using the appropriateisotopically labelled reagent or starting material instead of thecorresponding non-isotopically labelled reagent or starting material, orby conventional techniques known to those skilled in the art.

It should be noted that the radical positions on any molecular moietyused in the definitions may be anywhere on such moiety as long as it ischemically stable.

As used herein, the following terms have the meanings as defined below:

C_(m)-C_(n)alkyl used on its own or in composite expressions such asC_(m)-C_(n)haloalkyl, C_(m)-C_(n)alkylcarbonyl, C_(m)-C_(n)alkylamine,C_(m)-C_(n)alkylsulphonyl, C_(m)-C_(n)alkylsufonylamino etc. representsa straight or branched alkyl radical having the number of carbon atomsdesignated, e.g. C₁-C₄alkyl means an alkyl radical having from 1 to 4carbon atoms. Preferred alkyl radicals for use in the present inventionare C₁-C₄alkyl and includes methyl, ethyl, n-propyl, isopropyl, t-butyl,n-butyl and isobutyl. Methyl and t-butyl are typically preferred.C₁-C₆alkyl has a corresponding meaning, including also all straight andbranched chain isomers of pentyl and hexyl. Other recitals ofC_(m)-C_(n)alkyl, such as C₅-C₁₀ alkyl have the corresponding meaning.

The term Me means methyl, MeO means methoxy, Et means ethyl and Ac meansacetyl.

C₀-C₂alkylene used in composite expressions such asC₃-C₆cycloalkylC₀-C₂alkylene refers to a divalent radical derived from amethyl or ethyl group, or in the case of C₀ the term C₀-C₂alkylene meansa bond.

C₁-C₄haloalkyl refers to C₁-C₄alkyl, wherein at least one C atom issubstituted with a halogen, preferably chloro or fluoro. Trifluoromethylis typically preferred

C₁-C₄alkoxy represents a radical C₁-C₄alkyl-O wherein C₁-C₄alkyl is asdefined above, and includes methoxy, ethoxy, n-propoxy, isopropoxy,t-butoxy, n-butoxy and isobutoxy. Methoxy and isopropoxy are typicallypreferred. C₁-C₆alkoxy has a corresponding meaning, expanded to includeall straight and branched chain isomers of pentoxy and hexoxy. Otherrecitals of C_(m)-C_(n)alkoxy, such as C₅-C₁₀alkoxy have thecorresponding meaning.

C₁-C₄haloalkoxy as used herein is meant to include C₁-C₄alkoxy whereinat least one C-atom is substituted with one or more halogen atom(s),typically chloro or fluoro. In many cases trifluoromethyl is preferred.

C₁-C₄alkoxycarbonyl means a radical C₁-C₄alkyl-O—C(═O).

Carbocyclyl includes cyclopentyl, cyclohexyl and especially cyclopropyland cyclobutyl. Carbocyclyl further includes cyclopentenyl andcyclohexenyl, in each case with a single double bond. A frequentlypreferred value for Carbocyclyl is phenyl.

Cyclic amine includes aziridine, azetidine, pyrrolidine, piperidine,piperazine and morpholine.

Het is a stable, monocyclic or bicyclic, saturated, partially saturatedor aromatic ring system, containing 1-4 hetero atoms independentlyselected from O, S and N, and each ring having 5 or 6 ring atoms;Exemplary aromatic Het include furan, thiophene, pyrrole, imidazole,pyrazole, triazole, tetrazole, thiazole, oxazole, isoxazole, oxadiazole,thiadiazole, isothiazole, pyridine, pyridazine, pyrazine, pyrimidine,quinoline, isoquinoline, benzofuran, benzothiophene, indole, indazoleand the like. Exemplary unsaturated Het include tetrahydrofuran, pyran,dihydropyran, 1,4-dioxane, 1,3-dioxane, piperidine, pyrrolidine,morpholine, tetrahydrothiopyran, tetrahydrothiophene, 2-H-pyrrole,pyrroline, pyrazoline, imidazoline, thiazolidine, isoxazolidine and thelike.

The compounds of the invention include a number of handles such as OH,NH or COOH groups to which conventional prodrug moieties can be applied.Prodrugs are typically hydrolysed in vivo to release the parent compoundin the plasma, liver or intestinal wall. Favoured prodrugs are esters ofhydroxyl groups such as a phenolic hydroxyl group at R⁴, or aminefunctions such as a sulphonamide amine function. Preferredpharmaceutically acceptable esters include those derived from C₁-C₆carboxylic acids such as acetyl or pivaloyl or optionally substitutedbenzoic acid esters, preferably unsubstituted or substituted withsubstituents broadly as described for R^(1a), typically 1-3 halo (e.g.F), C₁-C₄alkyl (e.g. Me), C₁-C₄haloalkyl (e.g. CF₃) or C₁-C₄alkyloxy(e.g. MeO) groups. Favoured sulphonamide prodrugs include aminoacylsderived from C₁-C₆ carboxylic acids such as acetyl or pivaloyl oroptionally substituted benzoic acid esters, preferably unsubstituted orsubstituted with substituents broadly as described for variable R^(1a),typically 1-3 halo (e.g. F), C₁-C₄alkyl (e.g. Me), C₁-C₄haloalkyl (e.g.CF₃) or C₁-C₄alkyloxy (e.g. MeO) groups.

Unless otherwise mentioned or indicated, the chemical designation of acompound encompasses the mixture of all possible stereochemicallyisomeric forms, which said compound may possess. Said mixture maycontain all diastereomers and/or enantiomers of the basic molecularstructure of said compound. All stereochemically isomeric forms of thecompounds of the present invention both in pure form or mixed with eachother are intended to be embraced within the scope of the presentinvention.

Pure stereoisomeric forms of the compounds and intermediates asmentioned herein are defined as isomers substantially free of otherenantiomeric or diastereomeric forms of the same basic molecularstructure of said compounds or intermediates. In particular, the term“stereoisomerically pure” concerns compounds or intermediates having astereoisomeric excess of at least 80% (i.e. minimum 90% of one isomerand maximum 10% of the other possible isomers) up to a stereoisomericexcess of 100% (i.e. 100% of one isomer and none of the other), more inparticular, compounds or intermediates having a stereoisomeric excess of90% up to 100%, even more in particular having a stereoisomeric excessof 94% up to 100% and most in particular having a stereoisomeric excessof 97% up to 100%. The terms “enantiomerically pure” and“diastereomerically pure” should be understood in a similar way, butthen having regard to the enantiomeric excess, and the diastereomericexcess, respectively, of the mixture in question.

Compounds of the invention can be prepared as their individualstereoisomers by reacting a racemic mixture of the compound with anoptically active resolving agent to form a pair of diastereoisomericcompounds, separating the diastereomers and recovering the opticallypure enantiomer. While resolution of enantiomers can be carried outusing covalent diasteromeric derivatives of compounds of Formula I,dissociable complexes are preferred (e.g., crystalline;diastereoisomeric salts). Diastereomers have distinct physicalproperties (e.g., melting points, boiling points, solubilities,reactivity, etc.) and can be readily separated by taking advantage ofthese dissimilarities. The diastereomers can be separated bychromatography, for example HPLC or, preferably, byseparation/resolution techniques based upon differences in solubility.The optically pure enantiomer is then recovered, along with theresolving agent, by any practical means that would not result inracemization. A more detailed description of the techniques applicableto the resolution of stereoisomers of compounds from their racemicmixture can be found in Jean Jacques Andre Collet, Samuel H. Wilen,Enantiomers, Racemates and Resolutions, John Wiley & Sons, Inc. (1981).

While it is possible for the active agent to be administered alone, itis preferable to present it as part of a pharmaceutical formulation.Such a formulation will comprise the above defined active agent togetherwith one or more acceptable carriers/excipients and optionally othertherapeutic ingredients. The carrier(s) must be acceptable in the senseof being compatible with the other ingredients of the formulation andnot deleterious to the recipient.

The formulations include those suitable for rectal, nasal, topical(including buccal and sublingual), vaginal or parenteral (includingsubcutaneous, intramuscular, intravenous and intradermal)administration, but preferably the formulation is an orally administeredformulation. The formulations may conveniently be presented in unitdosage form, e.g. tablets and sustained release capsules, and may beprepared by any methods well known in the art of pharmacy. Such methodsinclude the step of bringing into association the above defined activeagent with the carrier. In general, the formulations are prepared byuniformly and intimately bringing into association the active agent withliquid carriers or finely divided solid carriers or both, and then ifnecessary shaping the product. The invention extends to methods forpreparing a pharmaceutical composition comprising bringing a compound ofFormula I or its pharmaceutically acceptable salt in conjunction orassociation with a pharmaceutically acceptable carrier or vehicle. Ifthe manufacture of pharmaceutical formulations involves intimate mixingof pharmaceutical excipients and the active ingredient in salt form,then it is often preferred to use excipients which are non-basic innature, i.e. either acidic or neutral.

Formulations for oral administration in the present invention may bepresented as discrete units such as capsules, cachets or tablets eachcontaining a predetermined amount of the active agent; as a powder orgranules; as a solution or a suspension of the active agent in anaqueous liquid or a non-aqueous liquid; or as an oil-in-water liquidemulsion or a water in oil liquid emulsion and as a bolus etc.

With regard to compositions for oral administration (e.g. tablets andcapsules), the term suitable carrier includes vehicles such as commonexcipients e.g. binding agents, for example syrup, acacia, gelatin,sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose,ethylcellulose, sodium carboxymethylcellulose,hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers,for example corn starch, gelatin, lactose, sucrose, microcrystallinecellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride andalginic acid; and lubricants such as magnesium stearate, sodium stearateand other metallic stearates, glycerol stearate stearic acid, siliconefluid, talc waxes, oils and colloidal silica. Flavouring agents such aspeppermint, oil of wintergreen, cherry flavouring or the like can alsobe used. It may be desirable to add a colouring agent to make the dosageform readily identifiable. Tablets may also be coated by methods wellknown in the art.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active agent in a free flowingform such as a powder or granules, optionally mixed with a binder,lubricant, inert diluent, preservative, surface-active or dispersingagent. Moulded tablets may be made by moulding in a suitable machine amixture of the powdered compound moistened with an inert liquid diluent.The tablets may be optionally be coated or scored and may be formulatedso as to provide slow or controlled release of the active agent.

Other formulations suitable for oral administration include lozengescomprising the active agent in a flavoured base, usually sucrose andacacia or tragacanth; pastilles comprising the active agent in an inertbase such as gelatine and glycerine, or sucrose and acacia; andmouthwashes comprising the active agent in a suitable liquid carrier.

As with all pharmaceuticals, the appropriate dosage for the compounds orformulations of the invention will depend upon the indication, theseverity of the disease, the size and metabolic vigour and the patient,the mode of administration and is readily determined by conventionalanimal trials. Dosages providing intracellular (for inhibition ofphysiological proteases of the papain superfamily) concentrations of theorder 0.01-100 μM, more preferably 0.01-10 μM, such as 0.1-5 μM aretypically desirable and achievable.

Compounds of the invention are prepared by a variety of solution andsolid phase chemistries.

A typical first step is the preparation of a P1 building block of theformula III

where R^(2a) and R^(2b) are as defined above and PG is a conventional Nprotecting group such as Boc, CBz or Fmoc. These building blocks arenovel and constitute a further aspect of the invention. The P1 buildingblock (III) is subsequently transformed into a P1-prime side unit offormula (IV)

where R^(2a) and R^(2b) are as defined above and PG is a conventional Nprotecting group such as Boc, CBz or Fmoc. These building blocks arenovel and constitute a further aspect of the invention.

Building blocks of formula III and IV are typically prepared asdescribed in scheme 1 below.

A suitable starting material is an N-protected cycloalkyl amino acid, ofwhich several are available commercially or can be prepared as shown inthe following Examples or as described by Allan et al. in J. Med. Chem.,1990 33 (10) 2905-2915.

The carboxylic acid (1a) is transformed via a Weinreb synthesis to aN,O-dimethylhydroxamic acid (1b) which provides the correspondingaldehyde (1c). The aldehyde may also be accessed by reduction of thecarboxylic function of a cycloalkyl amino acid followed by oxidationunder Dess Martin conditions. The aldehyde (1c) can be subsequentlyreacted with t.butyl isocyanide or equivalent in a Passerini reaction toafford the α-hydroxy amide (1d). Subsequent hydrolysis of the amide thenprovides the required α-hydroxycarboxylic acid P1 building block (III).

Generally the strongly acidic conditions required to hydrolyse the amidealso lead to loss of the NBoc protection, if used. Hence, the amine canbe used directly to couple to a P2 building block or else if it needs tobe stored, the amine can be reprotected. The P1 building block (III) isthen modified at the C terminus to provide the primary amide P1-primeside unit (IV). Typically the P1 building block (III) is transformed toa primary amide via the methyl ester by reaction with methyl iodide inthe presence of a suitable base, such as potassium carbonate or thelike, followed by hydrolysis of the intermediate methyl ester to theprimary amide effected for instance by treatment with a saturatedsolution of ammonia in methanol.

P1-prime side unit IV thus afforded is then extended at N terminus toprovide compounds of formula I. Scheme 2 below illustrates a route tocompounds of formula I wherein R⁴ is C₁-C₆alkyl, C₁-C₆haloalkyl, Het orCarbocyclyl.

The P1-prime side unit (IV) is deprotected at the N terminus andelongated with the P2 and subsequently P3 building blocks usingconventional peptide chemistries. For example a P2 residue can beintroduced via BocP2-OH using standard coupling conditions such as HATU,DIPEA in DMF. The terminal Boc protection is again removed with acetylchloride in methanol or equivalent and the P3 residue introduced viaP3-OH using standard coupling conditions such as HATU, DIPEA in DMF.Alternatively, A P3-P2-building block may be prepared separately andadded directly to the P1-prime side-building block (IV) in one step,thus affording the dipeptide derivative (2b). Oxidation of the α-hydroxyamide to the corresponding α-keto amide (2c) is then effected using anyconvenient oxidation method known in the art, such as Dess Martinoxidation or Moffat oxidation or the like.

Compounds of formula II, i.e. compounds wherein R⁴ is C₁-C₆alkoxy,C₁-C₆haloalkoxy, can be prepared from the N-protected amine 2a asgenerally illustrated in scheme 3.

Removal of the N-protecting group from the N-protected amine (2a) usingstandard techniques well known in the art of peptide synthesis, such astreatment with acid in the case of a boc-protecting group, followed byreaction with a suitable alkoxycarbonylating agent such as an alkylchloroformate or a dialkyl dicarbonate, optionally in the presence of abase such as diisopropylethyl amine or similar, provides the carbamate(3a). Oxidation of the α-hydroxy amide to the corresponding α-keto amide(3b) is then effected using any convenient oxidation method known in theart, such as Dess Martin oxidation or Moffat oxidation or the like.

An extensive range of appropriately protected L-amino acids suitable forP2 building blocks and carboxylic acids, carboxylic acid halides andcarbamoyl halides suitable for P3 building blocks are commerciallyavailable or accessed by simple chemistries or as shown in WO06/064286.The P3 and P2 building blocks may alternatively be coupled first andthen reacted with the P1-prime side unit.

Elongation is typically carried out in the presence of a suitablecoupling agent e.g., benzotriazole-1-yloxytrispyrrolidinophosphoniumhexafluorophosphate (PyBOP),O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl-uroniumhexafluorophosphate (HATU),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), or1,3-dicyclohexyl carbodiimide (DCC), optionally in the presence ofl-hydroxybenzotriazole (HOBT), and a base such asN,N-diisopropylethylamine, triethylamine, N-methylmorpholine, and thelike. The reaction is typically carried out at 20 to 30° C., preferablyat about 25° C., and requires 2 to 24 h to complete. Suitable reactionsolvents are inert organic solvents such as halogenated organic solvents(e.g., methylene chloride, chloroform, and the like), acetonitrile,N,N-dimethylformamide, ethereal solvents such as tetrahydrofuran,dioxane, and the like.

Alternatively, the above elongation coupling step can be carried out byfirst converting the P3/P2 building block into an active acid derivativesuch as succinimide ester and then reacting it with the P1 amine. Thereaction typically requires 2 to 3 h to complete. The conditionsutilized in this reaction depend on the nature of the active acidderivative. For example, if it is an acid chloride derivative, thereaction is carried out in the presence of a suitable base (e.g.triethylamine, diisopropylethylamine, pyridine, and the like). Suitablereaction solvents are polar organic solvents such as acetonitrile,N,N-dimethylformamide, dichloromethane, or any suitable mixturesthereof.

The term “N-protecting group” or “N-protected” as used herein refers tothose groups intended to protect the N-terminus of an amino acid orpeptide or to protect an amino group against undesirable reactionsduring synthetic procedures. Commonly used N-protecting groups aredisclosed in Greene, “Protective Groups in Organic Synthesis” (JohnWiley & Sons, New York, 1981), which is hereby incorporated byreference. N-protecting groups include acyl groups such as formyl,acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl,2-bromoacetyl, trifluoracetyl, trichloroacetyl, phthalyl,o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4-chlorobenzoyl,4-bromobenzoyl, 4-nitrobenzoyl, and the like; sulfonyl groups such asbenzenesulfonyl, p-toluenesulfonyl, and the like, carbamate forminggroups such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl,p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl,3,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl,2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl,t-butoxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl,ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl,fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, and thelike; alkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl andthe like; and silyl groups such as trimethylsilyl and the like. FavouredN-protecting groups include formyl, acetyl, benzoyl, pivaloyl,t-butylacetyl, phenylsulfonyl, benzyl (bz), t-butoxycarbonyl (BOC) andbenzyloxycarbonyl (Cbz).

Hydroxy and/or carboxy protecting groups are also extensively reviewedin Greene ibid and include ethers such as methyl, substituted methylethers such as methoxymethyl, methylthiomethyl, benzyloxymethyl,t-butoxymethyl, 2-methoxyethoxymethyl and the like, silyl ethers such astrimethylsilyl (TMS), t-butyldimethylsilyl (TBDMS) tribenzylsilyl,triphenylsilyl, t-butyldiphenylsilyl triisopropyl silyl and the like,substituted ethyl ethers such as 1-ethoxymethyl,1-methyl-1-methoxyethyl, t-butyl, allyl, benzyl, p-methoxybenzyl,dipehenylmethyl, triphenylmethyl and the like, aralkyl groups such astrityl, and pixyl (9-hydroxy-9-phenylxanthene derivatives, especiallythe chloride). Ester hydroxy protecting groups include esters such asformate, benzylformate, chloroacetate, methoxyacetate, phenoxyacetate,pivaloate, adamantoate, mesitoate, benzoate and the like. Carbonatehydroxy protecting groups include methyl vinyl, allyl, cinnamyl, benzyland the like.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Various embodiments of the invention will now be described by way ofillustration only with reference to the following Examples.

In the examples below, the following systems are typically employed:

Nuclear Magnetic Resonance (NMR) spectra were recorded on a VarianGemini 7 Tesla 300 MHz instrument, or a Bruker Avance 400 MHz instrumentin the solvent indicated. Chemical shifts are given in ppm down- andupfield from tetramethylsilane (TMS). Resonance multiplicities aredenoted s, d, t, m, br and app for singlet, doublet, triplet, multiplet,broad and apparent, respectively. The Mass Spectrometry (MS) spectrawere recorded on a Finnigan SSQ7000 TSP or a Finnigan SSQ710 DI/EIinstrument. LC-MS was obtained with a Waters 2790 LC-system equippedwith a Waters Xterra™ MS C₈ 2.5 μm 2.1×30 mm column, a Waters 996Photodiode Array Detector and a Micromass ZMD. High pressure liquidchromatography (HPLC) assays were performed using a Hewlett Packard 1100Series HPLC system equipped with a Zorbax column SB—C₈ 4.6 mm×15 cm.Column chromatography was performed using silica gel 60 (230-400 meshASTM, Merck) and thin layer chromatography (TLC) was performed on TLCprecoated plates, silica gel 60 F₂₅₄ (Merck).

Preparation of Building Block 1 (BB1), a P1 Building Block

Step a) 2-(1-((tert-butoxycarbonyl)amino)cyclobutyl)-2-hydroxyaceticacid (BB1-a)

To a solution of 1-tert-butoxycarbonylamino-cyclobutanecarboxylic acid(3 g, 13.94 mmol) in dry DMF (50 mL) was addedN,O-dimethylhydroxylamine×HCl (1.36 g, 13.94 mmol) and DIEA (9.21 mL,55.75 mmol). The reaction flask was cooled to 0° C. and after 10 minutesHATU (5.30 g, 13.94 mmol) was added to the solution (which turned yellowon addition). After 2 hrs the DMF was removed by rotary evaporation atreduced pressure. The residue was dissolved in EtOAc (100 mL) and washedtwice with 10% citric acid (aq) and saturated NaHCO₃ (aq) solution. Theorganic phase was dried with Na₂SO₄, filtered and evaporated on silica.The product was purified by flash chromatography (heptane:ethyl acetate(1:1) to give the product as a colourless oil that slowly crystallizes(3.13 g) in 87% yield.

Step b) (1-Formyl-cyclobutyl)-carbamic acid tert-butyl ester (BB 1-b)

LiAlH₄ (202 mg, 5.33 mmol) was added to a solution of the Weinreb amideBB1-a (1.10 g, 4.27 mmol) dissolved in dry diethyl ether (35 mL) at 0°C. The solution was stirred at 15 minutes before the reaction wasquenched with slow addition of potassium hydrogen tartaric acid (sat,aq) and stirred for 10 minutes. The solution was poured into aseparatory funnel and the water phase was extracted with ethyl acetatetwice. The combined organic phases were washed with 0.5 M HCl (3 times),NaHCO₃ (aq) (2 times) and brine (1 time). The organic phase was driedwith Na₂SO₄, filtered and evaporated on silica. The product was purifiedby flash chromatography (heptane:ethyl acetate (4:1→3:1) to give theproduct as white crystals (0.647 g) in 76% yield.

Step c) [tert-Butylcarbamoyl-hydroxy-methyl)-cyclobutyl]-carbamic acidtert-butyl ester (BB1-c)

BB1-b, (1.75 g, 8.78 mmol) was dissolved in CH₂Cl₂ (18 mL) and cooled inan ice bath, under inert gas. Pyridine (2.85 mL) was added, followed byt-butyl isocyanide (1.50 mL, 13.3 mmol). Trifluoroacetic acid (1.35 mL,17.5 mmol) was then added dropwise over 30 min. The yellow solution wasstirred at RT overnight. The mixture was concentrated, diluted withEtOAc (100 mL) and washed successively with 1N HCl (50 mL), saturatedNaHCO₃ (50 mL) and saturated NaCl (2×50 mL). Drying (Na₂SO₄) andconcentration under vacuum. The afforded crude product was treated withTHF (2.5 mL) and 1M LiOH in 3/1 MeOH-water (2.5 mL) at RT. TLC (3/1petroleum ether—EtOAc) showed complete ester hydrolysis after 15 min.After 45 min reaction time, 1N HCl (2.5 mL), water (10 mL) and EtOAc (20mL) were added, and the layers were separated. The organic phase waswashed with saturated NaHCO₃ (20 mL) and then saturated NaCl (2×20 mL),dried (Na₂SO₄) and concentrated. Flash chromatography (75 g silica, 5/1to 1/1 petroleum ether—EtOAc) gave a white solid (2.36 g, 89%).

Step d (1-tert-Butoxycarbonylamino-cyclobutyl)-hydroxy-acetic acid (BB1)

BB1-c (1.30 g, 4.33 mmol) was refluxed with 6N HCl (40 mL) until amidehydrolysis was complete as monitored by LCMS. The mixture wasevaporated, co-evaporating several times with water. 1M NaOH (15 mL) wasadded to the residue and the basic solution was stirred under vacuum for15 min. Boc₂O (1.92 g, 8.80 mmol) in dioxane (10 mL) was added, keepingpH at 10-11, and the mixture was stirred at RT overnight. The mixturewas diluted with water (50 mL), acidified with 1N HCl to pH 3, in an icebath, and then extracted with EtOAc (2×50 mL, then 30 mL). The organicphase was washed with saturated NaCl (50 mL), dried (Na₂SO₄) andevaporated to give crude P1 building block BB1 (0.649 g).

^(1H)NMR (400 MHz, d₆-DMSO) δ 6.88 (br s, 1H), 4.15 (s, 1H), 2.40 (br m,2H), 1.98 (br m, 2H), 1.80 (br m, 2H), 1.35 (s, 9H); ms ES⁺ m/z 146(100%), 190 (50%).

Preparation of Building Block 2 (BB2), a P1 Building Block

Step a) ((1-Bromo-3-chloropropan-2-yloxy)methyl)benzene (BB2-a)

To a stirred mixture of benzyl bromide (185 g, 1.08 mol) and (1.5 g) ofmercurous chloride was added epichlorohydrin (100 g, 1.08 mol). Thereaction mixture was heated for 12 hr at 100° C. TLC analysis confirmedformation of product. The product was separated from the dark brownreaction mixture by column chromatography using petroleum ether aseluent. TLC system; Petroleum ether:ethyl acetate (9:1), R_(F)=0.7.Yield; 148 g, 51%.

Step b) 3-Benzyloxy-cyclobutane-1,1-dicarboxylic acid diethyl ester(BB2-b)

To a stirred suspension of sodium hydride (22.5 g, 0.562 mol) in 800 mLof dry dioxane, was added diethyl malonate (90 g, 0.562 mol) drop-wiseover 20 min. After this addition was complete, BB2-a (148 g, 0.56 mol)was added drop-wise over 20 min. The mixture was then heated at refluxfor 24 hr. After cooling to room temperature, sodium hydride (22.5 g,0.562 mol) in a little dioxane (˜20 mL) was added to the mixture andheating at reflux was continued for an additional 48 hr. The solvent waspartially removed under reduced pressure and the mixture was treatedwith 800 mL of water. This mixture was then extracted with ethyl acetate(500 mL×3), extracts were dried (Na₂SO₄) and concentrated in vacuo andthe residue was purified by column chromatography using petroleumether:ethyl acetate (10%) which gave the title compound. TLC system;petroleum ether:ethyl acetate (9:1), R_(F)=0.3. Yield: 100 g, 58%

Step c) Diethyl 3-hydroxycyclobutane-1,1-dicarboxylate (BB2-c)

To a solution of compound BB2-b (40 g) in EtOH (500 mL) was added 10%palladium on charcoal (4 g) and the mixture was hydrogenated for 3.5hours at 50 psi at room temperature. The catalyst was removed byfiltration, washed with ethyl acetate, EtOH and the solvent was thenremoved under reduced pressure. The residue was purified by silica gelchromatography with hexane/ethyl acetate as eluent to provide the titlecompound. TLC system; Petroleum ether:ethyl acetate (9:1), R_(F)=0.3.Yield: 18 g, 64%.

Step d) Diethyl 3-oxocyclobutane-1,1-dicarboxylate (BB2-d)

To a solution of compound BB2-c (18 g, 0.0833 mol) in DCM (200 mL) wasadded PCC (37 g, 0.176 mol) and the mixture was stirred for four hoursat room temperature. The solution was filtered through a silica gelcolumn and the residue was washed with DCM/MeOH 98/2 and then filteredthrough a similar column. The combined fractions were evaporated underreduced pressure to provide the desired compound. TLC system; Petroleumether:ethyl acetate (9:1), R_(F)=0.3. Yield: 11 g, 62%.

Step e) Diethyl 3,3-difluorocyclobutane-1,1-dicarboxylate (BB2-e)

To a cooled solution of compound BB2-d (11 g, 0.0513 mol) in dry DCM(150 mL) was added drop-wise a solution of DAST (18.72 g, 0.116 mol) andthe mixture was stirred at room temperature overnight. The mixture wasadded to ice water and was extracted three times with DCM. The solutionwas dried with sodium sulphate and evaporated under reduced pressure.The residue was purified by silica gel chromatography employinghexane/ethyl acetate as eluent to provide the title compound. TLCsystem; Petroleum ether:ethyl acetate (7:3), R_(F)=0.5. Yield: 7.7 g,64%.

Step f) 1-(Ethoxycarbonyl)-3,3-difluorocyclobutanecarboxylic acid(BB2-f)

Compound BB2-e (7.7 g, 0.0325 mol) was dissolved in ice cooled 0.5 Methanolic potassium hydroxide solution (30 mL) and water (6 mL). Themixture was stirred at room temperature overnight. Water was added andmost of the ethanol was removed under reduced pressure. The mixture wasacidified with 2M HCl and extracted three times with ethyl acetate. Theorganic phase was dried with sodium sulphate and evaporated underreduced pressure to give the desired compound. TLC system:petroleumether:ethyl acetate (1:1), R_(F)=0.3. Yield: 5.8 g, 86%.

Step g) Ethyl1-(tert-butoxycarbonylamino)-3,3-difluorocyclobutanecarboxylate (BB2-g)

To a solution of compound BB2-f (5.8 g, 0.0273 mol) in dry dioxane (100mL) was added tert-butanol (24.4 mL), DPPA (7.87 g, 0.027 mol) and TEA(2.87 g, 0.0284 mol) and the mixture was refluxed for five hours. Ethylacetate (about 200 mL) was added and the organic phase was washed twicewith 5% citric acid and saturated sodium hydrogen carbonate. Thesolution was dried and evaporated under reduced pressure. The desiredproduct was isolated by silica gel chromatography with hexane/ethylacetate. TLC system; Petroleum ether:ethyl acetate (1:1), R_(F)=0.5.Yield: 4 g, 51.4%.

Step h) tert-Butyl 3,3-difluoro-1-(hydroxymethyl)cyclobutylcarbamate(BB2-h)

To a ice cooled solution of compound BB2-g (4 g, 0.0143 mol) in dry THF(100 mL) was slowly added a solution of 2M lithium borohydride (30 mL)and the mixture was allowed to warm up to room temperature. The mixturewas stirred for three hours at room temperature. Ice water and 5% citricacid were added and the mixture was extracted three times with DCM. Theorganic phase was dried (Na₂SO₄), filtered and evaporated under reducedpressure which gave the title compound. TLC system petroleum ether:ethylacetate (1:1), R_(F)=0.3. Yield: 3.1 g, 91%.

Step i) tert-Butyl 3,3-difluoro-1-formylcyclobutylcarbamate (BB2-i)

To a solution of compound BB2-h (3.1 g, 0.0130 mol) in dry DCM (100 mL)was added Dess Martin Period inane (19.9 g, 0.0470 mol) and the mixturewas stirred for three hours at room temperature. Ethyl acetate (200 mL)was added and the organic phase was washed twice with 10% sodiumthiosulphate solution, twice with 0.5 M NaOH and with brine. The organicphase was dried and evaporated under reduced pressure. The residue waspurified by silica gel chromatography with hexane/ethyl acetate aseluent which gave the title compound. TLC system; petroleum ether:ethylacetate (1:1), R_(F)=0.4. Yield: 2.7 g, 87%.

Step j) tert-Butyl1-(2-(tert-butylamino)-1-hydroxy-2-oxoethyl)-3,3-difluorocyclobutylcarbamate(BB2-j)

To a ice cooled solution of compound BB2-i (1.5 g, 0.0064 mol) in dryDCM (100 mL) was added tert-butylisocyanate (0.81 g, 0.009 mol) andpyridine (2.04 g, 0.027 mol). Trifluoroacetic acid (1.58 g, 0.015 mol)was added over a ten minutes period. The mixture was stirred for fivehours at room temperature. Ethyl acetate was added and the organic phasewas washed twice with 5% citric acid and brine. The organic phase wasevaporated and dissolved in dioxane (50 mL). 1M LiOH solution (100 mL)was added and the mixture was stirred overnight at room temperature. 5%Citric acid was added and the mixture was extracted three times withethyl acetate. The organic phase was washed with brine, dried (Na₂SO₄),filtered and evaporated under reduced pressure. The product was purifiedby silica gel chromatography with hexane/ethyl acetate as eluent. TLCsystem; Petroleum ether:ethyl acetate (1:1), R_(F)=0.4. Yield: 1.0 g,46%.

Step k)2-(1-(tert-Butoxycarbonylamino)-3,3-difluorocyclobutyl)-2-hydroxyaceticacid (BB2)

Compound BB2-j (1 g) was dissolved in 6N HCl (40 mL), and heated toreflux for 24 h after which TLC showed that the reaction had reachedcompletion. The reaction mixture was concentrated in vacuo and residuewas dissolved in THF; H₂O (7; 3, 50 mL), and TEA (1.8 mL, 0.012 mol) andBoc anhydride (2.6 g, 0.012 mol) were both added. The mixture wasstirred at RT for 8 h when TLC confirmed the reaction had reachedcompletion. The reaction mixture was concentrated in vacuo and theresidue was purified by column chromatography using 5% methanol inchloroform which gave the title compound. TLC system; MeOH:CHCl₃ (1:9),R_(F)=0.4. Yield: 0.6 g, 72%.

^(1H) NMR (400 MHz, d₆-DMSO) δ 7.30 (br s, 1H), 4.11 (s, 1H), 2.90 (brm, 2H), 2.61 (br m, 2H), 1.35 (s, 9H); ms ES⁺ m/z 281 (100%).

Preparation of Building Block 3 (BB3), a P1 Building Block

Step a) ((1-Bromo-3-chloropropan-2-yloxy)methyl)benzene (BB3-a)

A mixture of benzyl bromide (46.0 g, 0.269 mol) and epichlorohydrin(24.9 g, 0.269 mol) and mercurous chloride (0.04 g, 0.085 mmol) washeated for 12 h at 150° C. The crude product was purified by columnchromatography (silica gel 60-120 mesh, eluent 1% EtOAc in pet ether)which afforded the title compound as a viscous liquid (50 g, yield 70%).TLC system: 10% EtOAc in pet ether, R_(f)=0.6.

Step b) Diethyl 3-(benzyloxy)cyclobutane-1,1-dicarboxylate (BB3-b)

In a three-neck flask equipped with stirrer, additional funnel andreflux condenser was place NaH (4.6 g, 0.192 mol) in dry dioxane (150mL). To this stirred reaction mixture, diethyl malonate (30.75 g, 0.192mol) was added drop-wise over 30 min. After the addition was complete,compound BB3-a (50 g, 0.19 mol) was added drop-wise over a period of 30min. The reaction mixture was refluxed for 24 h. After cooling to roomtemperature, NaH (4.6 g, 0.192 mol) and dry dioxane (40 mL) was added tothe reaction mixture and further heated to reflux for another 48 h. Thesolvent was partially removed under reduced pressure and the mixture wastreated with water (150 mL). The product was extracted with diethylether (3×100 mL), the organic layer was washed with brine and dried overanhydrous Na₂SO₄. Solvent was concentrated in vacuum and the crudeproduct was purified by column chromatography (silica gel 60-120 mesh,eluent 2% EtOAc in pet ether) which afforded the title compound as aviscous liquid (33 g, yield 57%). TLC system: 15% EtOAc in pet ether,R_(f)=0.5.

Step c) Diethyl 3-hydroxycyclobutane-1,1-dicarboxylate (BB3-c)

To a solution of compound BB3-b (33 g, 0.108 mol) in EtOH (300 mL) wasadded 10% palladium on charcoal (10 g) and the mixture was hydrogenatedfor 48 h with 50 psi pressure at room temperature. The catalyst wasremoved by filtration through a celite bed and washed thoroughly withEtOAc. The solvent was removed under reduced pressure. The product waspurified by silica gel chromatography (silica gel 60-120 mesh, eluent20% EtOAc in pet ether) which afforded the title compound as a viscousliquid (12 g, yield 51%). TLC system: 30% EtOAc in pet ether, R_(f)=0.3.

Step d) Diethyl 3-fluorocyclobutane-1,1-dicarboxylate (BB3-d)

Compound BB3-c (0.8 g, 0.0037 mol) was dissolved in dry DCM (16 mL) andcooled to 0° C. DAST (1.8 g, 0.011 mol) was added drop-wise to the coldsolution. The reaction mixture was warmed to room temperature stirredfor 12 h. The reaction mixture was quenched with cold saturated NaHCO₃solution. The crude product was extracted with DCM (100 mL). The organiclayer was washed with 10% NaHCO₃ solution, water followed by brine anddried over anhydrous Na₂SO₄. Solvent was concentrated in vacuum and thecrude product was purified by column chromatography (silica gel 60-120mesh, eluent 1-2% EtOAc in pet ether) which afforded the title compoundas a pale yellow liquid (460 mg, yield 57%). TLC system: 10% EtOAc inpet ether, R_(f)=0.4.

Step e) 1-(Ethoxycarbonyl)-3-fluorocyclobutanecarboxylic acid (BB3-e)

Compound BB3-d (0.46 g, 0.0021 mol) was dissolved in ice cooled 0.5Mpotassium hydroxide solution in EtOH (4.2 mL) and water (1.4 mL). Themixture was stirred at room temperature overnight. Water was added andmost of the ethanol was removed under reduced pressure. The mixture wasacidified with 2N HCl and extracted with EtOAc (3×50 mL). The organicphase was dried over anhydrous Na₂SO₄. Solvent was concentrated invacuum to afford the crude title compound (0.35 g, crude) which was usedas such for the next step. TLC system: 50% EtOAc in pet ether,R_(f)=0.3.

Step f) Ethyl1-(tert-butoxycarbonylamino)-3-fluorocyclobutanecarboxylate (BB3-f)

To a solution of compound BB3-e (0.35 g, 0.0018 mol) in dry dioxane (6mL) was added tert-butanol (1.8 mL), diphenyl phophoryl azide (0.56 g,0.002 mol) and triethylamine (0.2 g, 0.002 mol) and the mixture wasrefluxed for 5 h. After completion of the reaction, EtOAc (60 mL) wasadded to the reaction mixture and the organic layer was washed with 5%citric acid (2×20 mL) followed by saturated NaHCO₃ (50 mL). The organicsolvent was evaporated under reduced pressure. To the residue EtOAc (100mL) was added and the organic layer was washed with brine and dried overanhydrous Na₂SO₄. Solvent was concentrated in vacuum and the crudeproduct was purified by column chromatography (silica gel 60-120 mesh,eluent 5-10% EtOAc in pet ether) which afforded the title compound aswhite crystals (0.27 g, yield 56%). TLC system: 20% EtOAc in pet ether,R_(f)=0.4.

Step g) tert-Butyl 3-fluoro-1-(hydroxymethyl)cyclobutylcarbamate (BB3-g)

To a ice cooled solution of compound BB3-f (0.27 g, 0.001 mol) in dryTHF (10 mL) was slowly added a solution of 2M lithium borohydride (2 mL,0.004 mol) and the mixture was allowed to warm up to room temperature.The mixture was stirred for 3 h at room temperature. The reactionmixture was quenched with ice water (2 mL) and 5% citric acid (5 mL) andthe crude product was extracted with DCM (2×50 mL). The organic layerwas washed with brine and dried over anhydrous Na₂SO₄. Solvent wasconcentrated in vacuum and the crude product was purified by columnchromatography (silica gel 60-120 mesh, eluent 15-18% EtOAc in petether) which afforded the title compound as white solid (90 mg, yield39%). TLC system: 50% EtOAc in pet ether, R_(f)=0.5.

Step h) tert-Butyl 3-fluoro-1-formylcyclobutylcarbamate (BB3-h)

To a degassed solution of compound BB3-g (90 mg, 0.0004 mol) in dry DCM(4.5 mL) was added Dess-Martin Periodinane (0.21 g, 0.0005 mol) and themixture was stirred for 3 h at room temperature. EtOAc (30 mL) was addedand the organic layer was washed with 10% sodium thiosulphate solution(2×10 mL), 0.5 M NaOH (20 mL) and with brine. The organic layer wasdried over anhydrous Na₂SO₄. Solvent was concentrated in vacuum and thecrude product was purified by column chromatography (silica gel 60-120mesh, eluent 10-15% EtOAc in pet ether) which afforded the titlecompound as a white crystalline solid (75 mg, yield 87%). TLC system:20% EtOAc in pet ether, R_(f)=0.4.

Step i) tert-Butyl1-(2-(tert-butylamino)-1-hydroxy-2-oxoethyl)-3-fluorocyclobutylcarbamate(BB3-i)

To an ice cooled solution of compound BB3-h (1.3 g, 0.0059 mol) in dryDCM (25 mL) was added tert-butyl isocyanide (0.75 g, 0.0089 mol) and drypyridine (2.6 mL). Trifluoroacetic acid (0.9 mL, 0.0118 mol) was addedover a period of ten minutes maintaining the temperature at 0° C. Thereaction mixture was slowly warmed to room temperature and stirred for16 h. EtOAc (50 mL) was added and the organic phase was washed twicewith 5% citric acid and brine. The organic phase was evaporated and thecrude product was dissolved in THF (25 mL). 1M LiOH solution in MeOH—H₂O(3:2 v/v) (2.6 mL) was added and the mixture was stirred for 2 h at roomtemperature. The reaction mixture was quenched with 5% citric acid andthe mixture was extracted with ethyl acetate (2×25 mL). The organiclayer was washed with brine and dried over anhydrous Na₂SO₄. Solvent wasevaporated in vacuum and to afford the title compound which was pureenough to be used in the next step (1.6 g, yield 84%). TLC system: 20%EtOAc in pet ether, R_(f)=0.3.

Step j)2-(1-(tert-Butoxycarbonylamino)-3-fluorocyclobutyl)-2-hydroxyacetic acid(BB3)

Compound BB3-i (1.6 g, 0.005 mol) was refluxed with 6N HCl (60 mL) for16 h until the amide hydrolysis was complete. The solvent was evaporatedunder reduced pressure and co-evaporated several times with water. Theproduct was dissolved in THF:H₂O (7:3 v/v, 50 mL), cooled to 0° C. andEt₃N (2.1 mL, 0.015 mol) was added followed by di-tert-butyl dicarbonate(2.18 g, 0.01 mol). The mixture was stirred at room temperatureovernight (pH was monitored in a regular interval and kept ˜11throughout the reaction). The reaction mixture was neutralized with 1NHCl and the product was extracted with EtOAc (2×50 mL). The organiclayer was washed with brine and dried over anhydrous Na₂SO₄. The solventwas evaporated under reduced pressure followed by purification by columnchromatography (silica gel 60-120 mesh, eluent 5% MeOH in CHCl₃) whichafforded the title P1 building block as a solid (0.65 g, yield 50%). TLCsystem: 30% MeOH in CHCl₃, R_(f)=0.3.

¹H NMR (400 MHz, d₆-DMSO) δ 7.01 (br s, 1H), 5.16 (br m, 1H), 4.97 (brm, 1H), 2.49 (br m, 5H), 1.36 (s, 9H); ms ES⁺ m/z 262 (100%).

Building Block 4 (BB4), a P1 Building Block

Step a) tert-butyl 1-(hydroxymethyl)-3-methoxycyclobutylcarbamate(BB4-a)

500 mg (1.51 mmol) of tert-butyl1-((tert-butyldimethylsilyloxy)methyl)-3-hydroxycyclobutylcarbamate(prepared by reduction of ethyl-1[[(tert-butyloxy)carbonyl]amino]-3-hydroxycyclobutane-1-carboxylate asdescribed in J. Med. Chem., 1990 33 (10) 2905-2915) and proton sponge(N,N,N′,N′ tetramethylnapthalene-1,8 diamine) (1.63 g, 6.04 mmol) weredissolved in DCM (18 mL), cooled down to 0° C., and 447 mg (3.02 mmol)of trimethyloxonium borontetrafluoride was added in one portion as asolid under vigorous stirring. The reaction mixture was stirred for 3 hand diluted with DCM (50 mL) and brine (20 mL), added under vigorousstirring. The organic phase was washed with sodium bicarbonate, brine,dried over sodium sulphate, evaporated and purified on short silicacolumn (DCM as an eluent). The resulting product was dissolved in THF (5mL), and a solution of tetrabutylammonium fluoride in THF (1M, 4.5 mL)was added, and the reaction was stirred at room temperature for 4.5 h.The reaction was monitored by TLC and once deemed to have reachedcompletion, it was absorbed onto silica and purified on silica(EtOAc-hexane 1:1 to neat EtOAc) to give the title compound (251 mg,72%). LC/MS 232 (M+1).

Step b) tert-Butyl 1-formyl-3-methoxycyclobutylcarbamate (BB4-b)

Alcohol BB4-a was dissolved in DCM (20 mL) and Dess-Martin reagent wasadded in one portion. The reaction was stirred for 2.5 hours. Once thereaction was deemed to have reached completion, it was diluted with 50mL of DCM and 20 mL of 10% Na₂S₂O₃ was added. The mixture was stirred,washed with sodium bicarbonate, brine, and the organic phase was driedover sodium sulphate. Purification on silica (EtOAc-hexane 1:1 to neatEtOAc) gave the title compound (500 mg, 59%).

Step c)[1-tert-Butylcarbamoyl-hydroxymethyl-3-methoxycyclobutyl]-carbamic acidtert-butyl ester (BB4-c)

Aldehyde BB4-b, 4.45 mmol, was dissolved in dry DCM (11 mL). Pyridine (2mL) was added under stirring conditions, followed by adding tert-butylisocyanide (6.68 mmol). The reaction was placed in an ice-bath and TFA(0.68 mL) was added dropwise during 20 min. The reaction mixture wasstirred overnight. The reaction was then deemed to have reachedcompletion and the solvent was removed under reduced pressure. Theresidue was redissolved in EtOAc and washed with 1 M HCl (2×), sodiumbicarbonate, brine, and the organic phase dried over sodium sulphate andevaporated. The remaining residue was dissolved in dioxane and stirredwith lithium hydroxide solution overnight and neutralized with citricacid. The product was extracted with EtOAc from the resulting solutionand purified on silica (EtOAc-hexane 1:3 to 1:1) which gave 850 mg ofthe title compound (58%) TLC: rf=0.61 (EtOAc:hexane 1:1).

Step d) (1-tert-Butoxycarbonylamino-3-methoxy-cyclobutyl)-hydroxy-aceticacid (BB4)

The amide BB4-c (850 mg, 2.57 mmol) was refluxed with 6N HCl (60 mL) for16 h until the amide hydrolysis was complete. The solvent was evaporatedunder reduced pressure and co-evaporated with water. The product wasdissolved in THF:H₂O (7:3 v/v, 50 mL), cooled to 0° C. and Et₃N (1.4 mL,10.2 mmol) was added followed by di-tert-butyl dicarbonate (2.25 g, 10.2mol). The mixture was stirred at room temperature overnight. Thereaction mixture was washed with EtOAc followed by acidifying to pH3with 1N HCl and extracted with EtOAc (2×50 mL). The organic layer waswashed with brine and dried over anhydrous Na₂SO₄. The solvent wasevaporated under reduced pressure which afforded the title compound as asolid (360 mg, yield 51%).

Building Block 5 (BB5), a P2 Building Block

Step a) 2-tert-Butoxycarbonylamino-3-chloro-propionic acid methyl ester(BB5-a)

A solution of triphenylphosphine (65.8 g, 0.251 mol)) andhexachloroethane (59.4 g, 0.251 mol) in dichloromethane (850 mL) wasadded in one portion to a solution of N-Boc-serine methyl ester (50 g,0.228 mol) in dichloromethane (170 mL) under argon atmosphere. Thereaction mixture was stirred at room temperature for 2 h and then thereaction was quenched with a saturated solution of NaHCO₃ (150 mL). Theorganic product was extracted with dichloromethane (2×300 mL) and thecombined organic layers was washed with brine (300 mL) and dried overanhydrous sodium sulphate. The solution was concentrated under reducedpressure and then triturated with Et₂O (500 mL). After filtration andevaporation of the solvent, the crude product was purified bychromatography on a silica column eluted with 6-8% EtOAc in pet. ether)which gave the title compound (42 g, 78%).

Step b) (2-Chloro-1-hydroxymethyl-ethyl)-carbamic acid tert-butyl ester(BB5-b)

Lithium borohydride (4.3 g, 0.195 mol) was added in portions to astirred solution of BB5-a (42 g, 0.177 mol) in EtOH-THF 9:1 at 0° C.under argon atmosphere. The reaction was stirred for 8 h at roomtemperature then quenched with a saturated solution of ammonium chloride(20 mL). The product was extracted with EtOAc (2×300 mL). The combinedorganic layers was washed with brine (300 mL) and dried over anhydroussodium sulphate. The solvent was evaporated under reduced pressure andthe afforded crude product was purified by chromatography on a silicacolumn eluted with 15% EtOAc in pet. ether, which gave the titlecompound (27.5 g, 74%).

Step c)[2-Chloro-1-(2-trimethylsilanyl-ethoxymethoxymethyl)-ethyl]-carbamicacid tert-butyl ester (BB5-c)

(2-Chloromethoxy-ethyl)-trimethyl-silane (26.18 g, 0.157 mol) was addeddrop-wise to a stirred solution of compound BB5-b (27.5 g, 0.131 mol)and N,N-diisopropylethylamine (27.4 mL, 0.157 mol) in dichloromethane(350 mL) at 0° C. under argon atmosphere. The reaction was allowed toattain room temperature and stirred for 18 h. The reaction mixture wasconcentrated under vacuum and then diluted with EtOAc (150 mL). Theproduct was extracted with EtOAc (2×200 mL) and the organic layer waswashed with brine (100 mL) and dried over anhydrous sodium sulphate. Thesolvent was evaporated under reduced pressure and the afforded crudeproduct was purified by chromatography on silica a column eluted with 5%EtOAc in pet. ether, which gave the title compound (25.5 g, 57%).

Step d)[2-(1-Hydroxy-cyclobutyl)-1-(2-trimethylsilanyl-ethoxymethoxymethyl)-ethyl]-carbamicacid tert-butyl ester (BB5-d)

n-BuLi (10 mL, 0.016 mol, 1.6 M solution in hexanes) was added drop-wiseto a stirred solution of compound BB5-c (2 g, 5.88 mmol) in THF (170 mL)at −78° C. under argon atmosphere. The stirring was continued for 15min, followed by drop-wise addition of LiNp (104 mL, 0.42 M solution inTHF, 0.044 mol) over 5 min. The dark solution was stirred at −78° C. for1 h and then cyclobutanone (0.88 mL, 11.77 mmol) was added drop-wise.The reaction mixture was stirred at −78° C. for 16 h then quenched witha saturated solution of NH₄Cl (50 mL) and allowed to warm to roomtemperature. The reaction was diluted with ether (100 mL) and asaturated solution of NH₄Cl (10 mL). The layers were separated and theaqueous layer was extracted with ether (2×100 mL). The combined organiclayers was dried (Na₂SO₄) and the solvent was evaporated under reducedpressure. The crude product was purified by chromatography on a silicacolumn eluted with heptane:ether 3:2, which gave the title compound(1.54 g, 70%).

Step e)[2-(1-Fluoro-cyclobutyl)-1-(2-trimethylsilanyl-ethoxymethoxymethyl)-ethyl]-carbamicacid tert-butyl ester (BB5-e)

BB5-d (0.5 g, 1.33 mmol), 50% Deoxofluor in THF (excess) and pyridine(excess) were mixed in DCM (10 mL). The resulting mixture was stirred atrt over night. The reaction mixture was washed with 10% citric acid (aq)and sat. NaHCO₃ (aq). The organic phase was dried (Na₂SO₄) andevaporated. The afforded crude product was purified by chromatography ona silica column using hexane:EtOAc (7:1 to 2:1) as eluent, which gavethe title compound (192 mg, 38%).

Step f) [2-(1-Fluoro-cyclobutyl)-1-hydroxymethyl-ethyl]-carbamic acidtert-butyl ester BB5-f)

A solution of BB5-e (192 mg, 0.51 mmol) in 0.1 M HCl in MeOH (20 mL) wasstirred for 3 hours, then triethylamine (1 mL) was added and thesolution was concentrated. The afforded crude product was purified bychromatography on a silica column using hexane:EtOAc (2:1) as eluent,which gave the title compound (69.3 mg, 55%) as a white solid.

Step g) 2-tert-Butoxycarbonylamino-3-(1-fluoro-cyclobutyl)-propionicacid (BB5)

BB5-f (69 mg, 0.279 mmol) and pyridine dichromate (1.15 g, 3.05 mmol)were dissolved in dry DMF (5 mL). After five hours H₂O (15 mL) was addedand the product was extracted with DCM (3×20 mL). The combined organiclayers was dried (Na₂SO₄) and evaporated. The crude was purified bychromatography on a silica column using hexane:EtOAc (1:1) followed byEtOH (100%) as eluent. This afforded the title compound as a white solid(22.3 mg, 31%), 262.4 [M+H]⁺.

Building Block 6 (BB6), a P1 α-Hydroxy Amide

Step a)(S)-tert-butyl(1-(2-((2,4-dimethoxybenzyl)amino)-1-hydroxy-2-oxoethyl)-3,3-difluorocyclobutyl)carbamate(BB6-a)

α-Hydroxy acid BB2 (1.5 g, 5.3 mmol) and 2,4-dimethoxy benzylamine (809mg, 5.35 mmol) was dissolved in CH₂Cl₂ (15 ml) and DMF (3 ml). HATU(2.066 g, 5.43 mmol) was added followed by diisopropylethylamine (948μl, 5.4 mmol) and the mixture was stirred at 22° C. over night. Themixture was diluted with CH₂Cl₂ and washed with 0.1 M HCl (aq)×2 andthen dried (Na₂SO₄), filtered and concentrated. The residue was purifiedby silica using p-ether/EtOAc (8:2→6:4) which gave the title compound(2.25 g, 5.22, 98% yield) mmol as a clear oil.

Step b) (S)-2-(1-amino-3,3-difluorocyclobutyl)-2-hydroxyacetamideHydrochloride salt (BB6)

Compound BB6-a (342 mg, 0.79 mmol) was dissolved in a solution ofTFA:CH₂Cl₂ 75:25 (3 mL ml) and 5% triethylsilane (150 μl). The mixturewas heated to 110° C. for 20 min in a micro wave reactor. The mixturewas then poured into an ice/1M HCl (aq) mixture and washed with CH₂Cl₂×3whereafter the acidic water phase was concentrated on a rotavapor at 35°C. which gave the title compound as hydrochloride salt [M+1]⁺=180.8

Building Block 7 (BB7), a P1 α-Hydroxyamide

(S)-2-(1-aminocyclobutyl)-2-hydroxyacetamide (BB7)

The title compound was prepared from α-hydroxy acid BB1 according to theprocedure described in BB6 steps a and b.

Building Block 8, a P1 α-Hydroxyamide

Step a)tert-butyl(1-(cyano((trimethylsilyl)oxy)methyl)cyclopentyl)carbamate(BB8-a)

To a solution of N-Boc-cycloleucinal (1.035 g, 4.85 mmol) in dry DCM (20ML) was added TMS-CN (645 μl, 4.85 mmol), and the solution was stirredfor 72 h, then quenched with saturated aq. NaHCO₃ and extracted withDCM. The organic layer was dried over Na₂SO₄ anhydrous, concentrated andthe residue purified using flash silica column with n-hexane:EtOAc 0-50%to give the title compound as a colourless oil (1.36 g). [M+1]⁺=312.8.

Step b) 2-(1-aminocyclopentyl)-2-hydroxyacetamide (BB8)

Cyano compound BB8-a (420.7 mg, 1.348 mmol) was dissolved in a solutionof formic acid:HCl 1:1 (6 mL) and the resulting solution was stirred for16 h. Acetonitrile was added and the solution concentrated, andco-evaporated with toluene to give the titled amine as hydrochloridesalt. [M+1]⁺=158.9.

Building Block 9, a P1 α-Hydroxyamide

2-(1-((Tert-butoxycarbonyl)amino)cyclopropyl)-2-hydroxyacetic acid (BB9)

The title compound was prepared in 29% overall yield fromN-Boc-1-aminocyclopropanecarboxylic acid (5.03 g, 25.0 mmol), accordingto the procedure described for the preparation of BB1.

Example C1

Step a)Tert-butyl(1r,3r)-1-(2-amino-1-hydroxy-2-oxoethyl)-3-fluorocyclobutylcarbamate(C1-a)

Potassium carbonate (147.5 mg, 1.06 mmol) was added to a solution of BB3(254 mg, 0.96 mmol) in DMF (5 mL) followed by addition of methyl iodide(72 μL, 1.15 mmol). The reaction mixture was stirred at room temperaturefor 2.5 h and then partitioned between DCM and aq. NaHCO₃ (sat.). Thephases were separated and the organic layer was washed with water, dried(Na₂SO₄) and concentrated. MS m/z 277.9 (M+H)⁺. The methyl ester cruderesidue formed was dissolved in a saturated solution of NH3 in methanol(10 mL) then heated at 60° C. for 5 days and concentrated which gave thetitle compound pure enough to be used in the next step without furtherpurification. MS m/z 262.9 (M+H)⁺.

Step b)Tert-butyl(S)-1-((1r,3S)-1-(2-amino-1-hydroxy-2-oxoethyl)-3-fluorocyclobutylamino)-4,4-dimethyl-1-oxopentan-2-ylcarbamate(C1-b)

The Boc-protected amine C1-a (0.48 mmol), was treated with HCl (4M indioxane, 5 mL) for 3 h whereafter the reaction mixture was concentrated.The resulting hydrochloride salt of the amine was added to a coldsolution of Boc-β-tBu-Ala-OH (129.5 mg, 0.53 mmol) and HATU (200 mg,0.53 mmol) in anh. DMF (4 mL) was added at 0° C., followed by additionof DIEA (335 μl, 1.92 mmol). The reaction mixture was stirred at roomtemperature over night, and then concentrated. Purification by flashcolumn chromatography (EtOAc/iso-Hexane, 0:1-7:3) gave the titlecompound (0.130 g, 70%). MS m/z 390.0 (M+H)⁺.

Step c)Tert-butyl(S)-1-((1r,3S)-1-(2-amino-2-oxoacetyl)-3-fluorocyclobutylamino)-4,4-dimethyl-1-oxopentan-2-ylcarbamate(C1)

Dess-Martin periodinane (0.301 g, 0.71 mmol) was added to a solution ofthe α-hydroxy amide C1b (0.184 g, 0.47 mmol) in DCM (5 ml). The reactionmixture was stirred at room temperature over night and quenched with 10%aq. Na₂S₂O₃ and aq. NaHCO₃ (sat.). The phases were separated and theorganic layer was dried (Na₂SO₄) and concentrated. Purification by flashcolumn chromatography (EtOAc/iso-Hexane, 0:1-1:0) gave the titlecompound (0.130 g, 67%). A small portion was further purified byRP-LC-MS (0.1% NH₄OH in acetonitrile-0.1% aq. NH₄OH, 40-65% over 9 min).MS m/z 386.5 (M−H)⁻.

Examples C2-C4

The compounds illustrated in the table below were prepared analogouslyto the procedure described in Example C1 using the appropriate P1 and P2building blocks, followed by oxidation to the end product α-keto amide.

TABLE C1

Ex. R³ R^(2a) R^(2b) [M + H]⁺ C2 2,2-dimethylbutyl H H 369.9 C3¹1-fluorocyclopentylmethyl F H 417.9 C4¹ 1-methylcyclopentylmethyl H H395.9 ¹Removal of the Boc group in step b was performed usingTFA/H₂O/TIS 9.5/0.25/0.25.

Example C5

[1-[1-(Carbamoyl-hydroxy-methyl)-3-fluoro-cyclobutylcarbamoyl]-2-(1-fluoro-cyclopentyl)-ethyl]-carbamicacid ethyl ester (C5-a)

Diisopropylethylamine (0.087 mL, 0.5 mmol) and ethyl chloroformate(0.024 mL, 0.25 mmol) were added to a stirred solution of the amineA1-b,2-amino-N-[1-(carbamoyl-hydroxy-methyl)-3-fluoro-cyclobutyl]-3-(1-fluorocyclopentyl)-propionamide(0.23 mmol) in DMF. After 15 min sat. aq. NaHCO₃ and DCM were added andthe organic layer was collected and concentrated and purified on flashsilica gel column using DCM-MeOH 0-10% which gave the title compound (62mg) [M+1]⁺ 391.9.

[1-(1-Aminooxalyl-3-fluoro-cyclobutylcarbamoyl)-2-(1-fluoro-cyclopentyl)-ethyl]-carbamicacid ethyl ester (C5)

A solution of Dess Martin periodinane (75 mg, 0.177 mmol) indichloroethane (5 mL) was added to solid[1-[1-(carbamoyl-hydroxy-methyl)-3-fluoro-cyclobutylcarbamoyl]-2-(1-fluoro-cyclopentyl)-ethyl]-carbamicacid ethyl ester (62 mg). After stirring for 2 h, NaHCO₃ (aq) was added,the layers were separated and the organic layer was concentrated. Theafforded crude product was purified on a prep RP HPLC-UV/MS, which gavethe title compound (14 mg). [M+1]⁺ 389.9.

Example C6

Step a) 2-Ethoxycarbonylamino-3-(1-fluorocyclopentyl)-propionic acid(C6-a)

2-tert-Butoxycarbonylamino-3-(1-fluorocyclopentyl)-propionic acid (200mg, 0.73 mmol) was kept in 4M HCl in dioxane (3 mL) for 20 min. Themixture was then freeze-dried and left under a vacuum for 2 h. The solidresidue was dissolved in dioxane-H₂O 1:1 (12 mL). NaHCO₃ (2.18 mmol, 3eq.) and then ethyl chloroformate (0.87 mmol 1.2 eq.) dissolved in ofdioxane (1 mL) were added. The reaction mixture was stirred for 20 minuntil completed according LC-MS. The mixture was acidified by citricacid (10% aq.) to pH 5. The product was extracted with DCM (20 mL) andEtOAc (20 mL). The organic layers were combined, dried (MgSO₄) andconcentrated. The afforded crude was used without further purificationin the next step.

Step b)[1-[1-(Carbamoyl-hydroxy-methyl)-cyclobutylcarbamoyl]-2-(1-fluorocyclopentyl)-ethyl]-carbamicacid ethyl ester (C6-b)

HATU (84 mg, 0.22 mmol) and diisopropylethylamine (0.14 mL, 0.8 mmol)was added to a stirred and cooled (0° C.) solution of2-ethoxycarbonylamino-3-(1-fluoro-cyclopentyl)-propionic acid (50 mg,0.2 mmol) in DMF. After 5 min, crude HCl salt of2-(1-amino-cyclobutyl)-2-hydroxy acetamide (290 mg, achieved from BB1 byremoval of the Boc group) was added and the temperature raised toambient. After 1 h sat. aq. NaHCO₃ and DCM were added and the organiclayer collected, concentrated and purified on a flash silica columnusing DCM-MeOH 0-10% which gave the title compound (37 mg). [M+1]⁺373.8.

Step c)[1-(1-Aminooxalyl-cyclobutylcarbamoyl)-2-(1-fluorocyclopentyl)-ethyl]-carbamicacid ethyl ester (C6)

The α-hydroxy amide C6-b (42 mg) was oxidized using Dess Martinperiodinane as described in Ex. C5, which gave the title compound (1.7mg). [M+1]⁺=371.9.

Example C7

(S)-ethyl(1-((1-(2-amino-2-oxoacetyl)cyclopentyl)amino)-3-(1-fluorocyclopentyl)-1-oxopropan-2-yl)carbamate(C7)

BB8 was coupled to the P2-building block(S)-2-((tert-butoxycarbonyl)amino)-3-(1-fluorocyclopentyl)propanoic acidas described in Ex. C1 step b whereafter the P3 moiety was added byreaction with ethyl chloroformate as described in Ex. C5, which gave thetitle compound. [M+1]⁺=386.8.

Example C8

Step a)(S)-tert-butyl(1-((1-(2-amino-2-oxoacetyl)-3,3-difluorocyclobutyl)amino)-3-(1-fluorocyclopentyl)-1-oxopropan-2-yl)carbamate(C8)

The amine BB6 was coupled to the P2-building block(S)-2-((tert-butoxycarbonyl)amino)-3-(1-fluorocyclopentyl)propanoic acidaccording to the method described in Ex. C1 step b. The affordedα-hydroxy amide (92 mg, 0.21 mmol) was dissolved in THF (2 mL) and DMSO(150 μL, 2.1 mmol) was added followed by EDC hydrochloride salt (201 mg,1 mmol). The solution was stirred for 15 min whereafter dichloroaceticacid (20 μL, 0.15 mmol) was added. The solution was stirred for 16 h,then diluted with DCM, and washed with aq. NaHCO₃ followed by citricacid (2% aq). The organic layer was separated and concentrated todryness and the residue purified using flash silica column with DCM-MeOH0-3% and further purified by prep LC-MS which gave the title compound (3mg). [M+1]⁺=436.18.

The compounds in Table C2 were prepared according to the proceduredescribed in Ex. C1, using the appropriate building blocks.

TABLE C2

Ex. R³ n [M + H]⁺ C9 cyclohexylmethyl 2 396.0 C101-fluorocyclopentylmethyl 2 400.4 C11 1-fluorocyclopentylmethyl 1 386.4C12 1-methylcyclopentylmethyl 1 382.4 C13 1-fluorocyclopentylmethyl 3413.9 ¹Removal of the Boc group in step b was performed usingTFA/H₂O/TIS 9.5/0.25/0.25.

Compound C14 was prepared according to the procedure described in Ex.C5, using the appropriate building blocks.

Example A1

Step a)tert-butyl((S)-1-(((1r,3S)-1-((S)-2-amino-1-hydroxy-2-oxoethyl)-3-fluorocyclobutyl)amino)-3-(1-fluorocyclopentyl)-1-oxopropan-2-yl)carbamate(A1-a)

The title compound was prepared from the Boc-protected hydroxy amineC1-a by reaction with the “P2-building block”(S)-2-((tert-butoxycarbonyl)amino)-3-(1-fluorocyclopentyl)propanoicacid, according to the method described in Ex. C1 step b.

Step b)2-Amino-N-[1-(carbamoyl-hydroxy-methyl)-3-fluoro-cyclobutyl]-3-(1-fluoro-cyclopentyl)-propionamide(A1-b)

The Boc-protected amine A1-a (0.223 mmol, 93 mg) was kept in a solutionof dichloromethane containing 20% TFA/water/triisopropylsilane9.5:0.25:0.25 (4 mL) for 30 min and then the solution was concentratedto dryness. DCE and 4M HCl in dioxane (1 mL) was added followed byconcentration to dryness, which gave the title compound as thehydrochloride salt. [M+1]⁺=320.1.

Step c)N-[1-[1-(Carbamoyl-hydroxy-methyl)-3-fluoro-cyclobutylcarbamoyl]-2-(1-fluoro-cyclopentyl)-ethyl]-3-imidazol-1-yl-benzamide(A1-c)

To a stirred solution of the “P3-acid” 3-imidazol-1-yl-benzoic acid (47mg, 0.25 mmol) in DMF (4 mL) was added HATU (95 mg, 0.25 mmol) anddiisopropylamine (0.147 mL, 1 mmol). After 2 min the amine A1-b (0.22mmol) was added. After 2 h a saturated aqueous solution of NaHCO₃ (3 mL)was added and the mixture was extracted with DCM (5 mL). The organicphase was concentrated and the residue purified using flash silicacolumn with DCM-MeOH 0-10%, which gave the title compound (10 mg).[M+1]⁺=489.9.

Step d)N-[1-(1-Aminooxalyl-3-fluoro-cyclobutylcarbamoyl)-2-(1-fluoro-cyclopentyl)-ethyl]-3-imidazol-1-yl-benzamide(A1)

A solution of Dess Martin periodinane in dichloromethane (5 mL) wasadded to the α-hydroxy amide A1-c (10 mg). After stirring for 2 h,NaHCO₃ (aq) was added, the layers were separated and the organic layerwas concentrated. The afforded crude product was purified on a prep RPHPLC-UV/MS, which gave the title compound (1.5 mg). [M+1]⁺ 487.8.

Example A2

Step a) Cyclopropanecarboxylic acid[1-[1-(carbamoyl-hydroxy-methyl)-cyclobutylcarbamoyl]-2-(1-methyl-cyclopentyl)-ethyl]-amide(A2-a)

The Boc-protected amine C4-b (40 mg, 0.1 mmol) was kept indichloromethane containing 20% TFA/water/triisopropylsilane9.5:0.25:0.25 (4 mL) for 2 h whereafter the solution was evaporated andthe residue put under vacuum for 2 h. The afforded free amine wasdissolved in DMF (4 mL) and diisopropylethylamine (0.052 mL, 0.3 mmol)was added, during stirring, followed by the addition ofcyclopropanecarbonyl chloride (0.012 mL, 0.12 mmol). aq. NaHCO₃ wasadded and the product extracted with dichloromethane. A flash silicacolumn in DCM-MeOH 0-10% gave the title compound (30 mg). [M+1]⁺=365.9.

Step b) Cyclopropanecarboxylic acid[1-(1-aminooxalyl-cyclobutylcarbamoyl)-2-(1-methyl-cyclopentyl)-ethyl]-amide(A2)

The α-hydroxy amide A2-a (30 mg, 0.08 mmol) was oxidized using DessMartin periodinane as described in Ex. C5 above. The afforded crudeproduct was purified on a prep RP HPLC-UV/MS which gave the titlecompound (5.7 mg). [M+1]⁺=363.9.

Examples A3-A4

Compounds A3 and A4 in the table below were prepared according to theprocedure outlined in Example A2, using the appropriate acid chloride.

TABLE A1

Ex. A3

Ex. A4

The compounds in the table below were prepared according to theprocedure outlined in Example A1, using the appropriate “P3-acid”R⁴C(═O)OH, “P1- and P2-building blocks”.

TABLE A2

Ex. R⁴ R³ R^(2a) R^(2b) [M + H]⁺ A5¹ cyclopropyl 2,2-dimethylbutyl F H356.1 A6¹ cyclopropyl (1-fluorocyclopentyl)methyl H H 368.2 A7^(1,2)2-methylpyridin-5-yl (1-fluorocyclopentyl)methyl H H 419.2 A8^(1,2)1,1,1-trifluoro-2- (1-fluorocyclopentyl)methyl H H 440.2hydroxypropan-2-yl A9^(1,2) 2-fluoropropan-2-yl 2,2-dimethylbutyl F F393.8 A10^(1,2) 1,1-difluoroethyl 2,2-dimethylbutyl F F 397.8 A11^(1,2)2-fluoropropan-2-yl (1-fluorocyclopentyl)methyl F F  424.16 A12^(1,2)1,1-difluoroethyl (1-fluorocyclopentyl)methyl F F 428.4 A13^(2,3)2-fluoropropan-2-yl 2,2-dimethylbutyl H H 358.0 A14^(2,3)1,1,1-trifluoro-2-methylpropan-2-yl 2,2-dimethylbutyl H H 408.0A15^(2,3) 2-fluoropropan-2-yl (1-methylcyclopentyl)methyl H H 384.0A16^(3,4) 2-methylprop-2-yl (1-fluorocyclopentyl)methyl H H 384.0A17^(1,5) 2,4-difluorophenyl (1-fluorocyclopentyl)methyl H H 440.4A18^(1,5) 3,4-difluorophenyl (1-fluorocyclopentyl)methyl H H 440.2A19^(1,5) thiazol-5-yl (1-fluorocyclopentyl)methyl H H 411.3 A20^(1,5)3,5-difluorophenyl (1-fluorocyclopentyl)methyl H H 440.4 A21^(1,5)2,4,6-trifluorophenyl (1-fluorocyclopentyl)methyl H H 458.4 A22^(1,5)3,4,5-trifluorophenyl (1-fluorocyclopentyl)methyl H H 458.4 A23^(1,5)2-fluoroprop-2-yl 1-fluorocyclopentylmethyl H H 388.4 A24²1,1,1-trifluoro-2-methylpropan-2-yl 1-methylcyclopentylmethyl H H 434.0A25 1-(trifluoromethyl)cyclopropyl 1-fluorocyclopentylmethyl H H 436.4A26 1-methylcyclopropyl 1-fluorocyclopentylmethyl H H 382.5 A271,1,1-trifluoro-2-methylpropan-2-yl 1-fluorocyclopentylmethyl H H 438.4A28 1-(trifluoromethyl)cyclobutyl 1-fluorocyclopentylmethyl H H 450.4A29 1,1,1-trifluoro-2-methylpropan-2-yl 1-methylcyclopentylmethyl F H454.3 A30 2-fluoropropan-2-yl 1-fluorocyclopentylmethyl F H 406.6 A312-fluoropropan-2-yl 1-methyl-cyclopentylmethyl F F 420.5 A321,1,difluoro-ethyl 1-methylcyclopentylmethyl F F 424.5 A331,1-difluoroethyl 1-fluorocyclopentylmethyl H H 392.0 A34 pyridin-2-yl1-fluorocyclopentylmethyl H H 405.0 ¹Removal of the Boc-group in step bwas performed using 4M HCl in dioxane. ²Oxidation of the α-hydroxy amideto the corr. α-keto amide was performed using DMSO-method described inEx. C8. ³Removal of the Boc-group in step b was performed with TFA inDCM. ⁴Oxidation of the α-hydroxy amide to corr. α-keto amide wasperformed using Dess Martin periodinane as described in Ex. C1.⁵Oxidation of the α-hydroxy amide to the corr. α-keto amide wasperformed by treating a solution of the alcohol with 15% solution ofDess-Martin periodinane in DCM.

The compounds in Table A3 below were prepared according to the proceduredescribed in Ex. A1, using the appropriate “P3-acid” R⁴C(═O)OH and “P1-and P2-building” blocks, and finally oxidized using Dess Martinperiodinane.

TABLE 3

Ex. R⁴ R^(3′) n [M + H]⁺ A35⁴ cyclopropyl F 3 382.08 A36⁴1,1,1-trifluoro-2-methylpropan-2-yl F 3 451.9  A37⁴ 3-methyloxetan-3-ylF 3 411.9  A38 1-(trifluoromethyl)cyclopropyl F 1 422.4  A391,1,1-trifluoro-2-methylpropan-2-yl F 1 424.4  A401-(trifluoromethyl)cyclopropyl CH₃ 1 418.3  A41² 3-methyloxetan-3-yl CH₃1 380.1  ²Oxidation of the α-hydroxy amide to the corr. α-keto amide wasperformed using DMSO-method described in Ex. C8. ⁴Oxidation of theα-hydroxy amide to corr. α-keto amide was performed using Dess Martinperiodinane as described in Ex. C1.

Example A42

Step a) methyl2-(1-((S)-2-((tert-butoxycarbonyl)amino)-3-(1-methylcyclopentyl)propanamido)cyclopropyl)-2-hydroxyacetate(A42-a)

The hydroxyl acid BB8 (100 mg, 0.43 mmol, 1 eq.) was dissolved inmethanol (10 mL) and TSM-CH₂N₂ (2M in hexanes) was added until theyellow colour remained and TLC showed no reaming starting material. Thesolvent was removed under vacuum. The remaining solid was dissolved inmethanol:acetic chloride (9:1) and resulting mixture was stirred at roomtemperature over night. The solvent was removed under vacuum. Theresidue was dissolved in DMF (5 mL), and2-tert-Butoxycarbonylamino-3-(1-methyl-cyclopentyl)-propionic acid (1eq.) and diisopropylethylamine (4 eq.) was added. Thereafter HATU (1eq.) was added and the resulting reaction mixture stirred at roomtemperature for 1.5 hours. The solvent was removed under vacuum and thecrude product was purified on silica column using hexane:EtOAc (3:3 to1:1) as eluent which gave the title compound (38%).

Step b)N-((2S)-1-((1-(2-amino-1-hydroxy-2-oxoethyl)cyclobutyl)amino)-3-(1-methylcyclopentyl)-1-oxopropan-2-yl)-2,2-difluorocyclopropanecarboxamide(A42-b)

Methyl 2-(1-((S)-2-((tert-butoxycarbonyl)amino)-3-(1methylcyclopentyl)-propanamido)cyclopropyl)-2-hydroxyacetate wasdissolved in methanol:acetic chloride (9:1) and resulting mixture wasstirred at room temperature over night. The solvent was removed undervacuum. The residue was dissolved in DMF (5 mL), and2,2-difluorocyclopropane-carboxylic acid (1 eq) anddiisopropylethylamine (4 eq) was added. Thereafter HATU (1 eq) was addedand the resulting reaction mixture stirred at room temperature for 1.5hours. The solvent was removed under vacuum and the crude product wasdissolve in EtOAc and washed with NaHCO3 (aq), HCL (1.2 M) and H₂O. Theorganic phase was concentrated under vacuum and the residue was dissolvein methanol. The mixture was cooled to 0° C. and NH₃ bubbled thru thereaction mixture. The solvent was evaporated and the crude product usedin next step.

N—((S)-1-((1-(2-amino-2-oxoacetyl)cyclopropyl)amino)-3-(1-methylcyclopentyl)-1-oxopropan-2-yl)-2,2-difluorocyclopropanecarboxamideStep c)(S)—N-(1-((1-(2-amino-2-oxoacetyl)cyclobutyl)amino)-3-(1-methylcyclopentyl)-1-oxopropan-2-yl)cyclopropanecarboxamideA42-1 & A42-2

A solution of Dess-Martin periodinane (1.5 eq) in dichloroethane (4 mL)was added to the solid α-hydroxy amide A42-b. The mixture was stirredfor 1 h and then NaHCO₃ (aq) was added. The organic layer was separatedand concentrated. Purification on prep RP HPLC-UV/MS gave the titlecompound as two separated diastereomers. [M+1]⁺ 386.3 and. [M+1]⁺ 386.3.

The below compounds were prepared according to the method described inExample A42, using the appropriate building blocks.

The compounds in the table below were prepared according to theprocedure outlined in Example A1, using the appropriate “P3-acid”R⁴C(═O)OH, “P1- and P2-building blocks”.

[M + Ex. R⁴ R³ R^(2a) R^(2b) H]⁺ A45 1,1-difluoroethyl1-fluorocyclopentylmethyl F H 410.3 A46 1,1-difluoroethyl1-methylcyclopentylmethyl F H 406.4 A47 3-methylpyridin-2-yl1-fluorocyclopentylmethyl H H 419.4 A48 2-fluoropropan-2-yl1-fluorocyclopentylmethyl F H 402.4 A49¹ 1,1,1.trifluoro-2-1-fluorocyclopentylmethyl OMe H 468.6 methylpropan-2-yl A50¹1,1-difluoroethyl 1-fluorocyclopentylmethyl OMe H A51¹2-fluoropropan-2-yl 1-fluorocyclopentylmethyl OMe H ¹Removal of theBoc-group in steps a and b was performed using conc. HCl:dioxane 1:3.

Example U1

S)—N-(1-(2-amino-2-oxoacetyl)cyclobutyl)-2-(3-ethylureido)-3-(1-methylcyclopentyl)-propanamide(U1)

The amine BB7 was coupled to the “P2-building block(S)-2-((tert-butoxycarbonyl)amino)-3-(1-methylcyclopentyl)propanoic acidaccording to the method described in Ex. C1 step b. The afforded amine(160 mg, 0.4 mmol) was kept in dichloromethane containing 20%TFA/water/triisopropylsilane 9.5:0.25:0.25 (8 mL) for 2 h whereafter thesolution was evaporated and the residue put under vacuum for 2 h. Theafforded free amine was dissolved in DMF (15 mL) anddiisopropylethylamine (0.21 mL, 1.2 mmol) was added, during stirring,followed by the addition of ethlyisocyanate (0.4 mmol, 0.032 mL). aq.NaHCO₃ was added and the product extracted with dichloromethane. A flashsilica column in DCM-MeOH 0-10% gave a residue to which was added to asolution of Dess-Martin periodinane (200 mg, 0.47 mmol) indichloroethane. After stirring for 2 h the solution was washed with aq.NaHCO₃, concentrated to dryness and purified on a prep. HPLC-UV/MS whichgave 24 mg of the title compound. [M+1]⁺=367.1.

Biological Examples Determination of Cathepsin K Proteolytic CatalyticActivity

Convenient assays for cathepsin K are carried out using humanrecombinant enzyme, such as that described in PDB.

ID BC016058 standard; mRNA; HUM; 1699 BP.DE Homo sapiens cathepsin K (pycnodysostosis), mRNA (cDNA cloneMGC:23107

RX MEDLINE; RX PUBMED; 12477932. DR RZPD; IRALp962G1234. DR SWISS-PROT;P43235;

The recombinant cathepsin K can be expressed in a variety ofcommercially available expression systems including E. coli, Pichia andBaculovirus systems. The purified enzyme is activated by removal of theprosequence by conventional methods.

Standard assay conditions for the determination of kinetic constantsused a fluorogenic peptide substrate, typically H-D-Ala-Leu-Lys-AMC, andwere determined in either 100 mM Mes/Tris, pH 7.0 containing 1 mM EDTAand 10 mM 2-mercaptoethanol or 100 mMNa phosphate, imM EDTA, 0.1%PEG4000 pH 6.5 or 100 mM Na acetate, pH 5.5 containing 5 mM EDTA and 20mM cysteine, in each case optionally with 1M DTT as stabiliser. Theenzyme concentration used was 5 nM. The stock substrate solution wasprepared at 10 mM in DMSO. Screens were carried out at a fixed substrateconcentration of 60 μM and detailed kinetic studies with doublingdilutions of substrate from 250 μM. The total DMSO concentration in theassay was kept below 3%. All assays were conducted at ambienttemperature. Product fluorescence (excitation at 390 nm, emission at 460nm) was monitored with a Labsystems Fluoroskan Ascent fluorescent platereader. Product progress curves were generated over 15 minutes followinggeneration of AMC product.

Cathepsin S Ki Determination

The assay uses baculovirus-expressed human cathepsin S and theboc-Val-Leu-Lys-AMC fluorescent substrate available from Bachem in a 384well plate format, in which 7 test compounds can be tested in parallelwith a positive control comprising a known cathepsin S inhibitorcomparator.

Substrate Dilutions

280 μL/well of 12.5% DMSO are added to rows B-H of two columns of a 96deep well polypropylene plate. 70 μL/well of substrate is added to rowA. 2×250 μL/well of assay buffer (100 mM Na phosphate, 100 mM NaCl, pH6.5) is added to row A, mixed, and double diluted down the plate to rowH.

Inhibitor Dilutions

100 μL/well of assay buffer is added to columns 2-5 and 7-12 of 4 rowsof a 96 well V bottom polypropylene plate. 200 μL/well of assay bufferis added to columns 1 and 6.

The first test compound prepared in DMSO is added to column 1 of the toprow, typically at a volume to provide between 10 and 30 times theinitially determined rough K_(i). The rough K_(i) is calculated from apreliminary run in which 10 μL/well of 1 mM boc-VLK-AMC ( 1/10 dilutionof 10 mM stock in DMSO diluted into assay buffer) is dispensed to rows Bto H and 20 μl/well to row A of a 96 well Microfluor™ plate. 2 μl ofeach 10 mM test compound is added to a separate well on row A, columns1-10. Add 90 μl assay buffer containing 1 mM DTT and 2 nM cathepsin S toeach well of rows B-H and 180 μl to row A. Mix row A using amultichannel pipette and double dilute to row G. Mix row H and read inthe fluorescent spectrophotometer. The readings are Prism data fitted tothe competitive inhibition equation, setting S=100 μM and K_(M)=100 μMto obtain an estimate of the K_(i), up to a maximum of 100 μM.

The second test compound is added to column 6 of the top row, the thirdto column 1 of the second row etc. Add 1 μL of comparator to column 6 ofthe bottom row. Mix column 1 and double dilute to column 5. Mix column 6and double dilute to column 10.

Using an 8-channel multistepping pipette set to 5×10 μL, distribute 10μL/well of substrate to the 384 well assay plate. Distribute the firstcolumn of the substrate dilution plate to all columns of the assay platestarting at row A. The tip spacing of the multichannel pipette willcorrectly skip alternate rows. Distribute the second column to allcolumns starting at row B.

Using a 12-channel multistepping pipette set to 4×10 μL, distribute 10μL/well of inhibitor to the 384 well assay plate. Distribute the firstrow of the inhibitor dilution plate to alternate rows of the assay platestarting at A1. The tip spacing of the multichannel pipette willcorrectly skip alternate columns. Similarly, distribute the second,third and fourth rows to alternate rows and columns starting at A2, B1and B2 respectively.

Mix 20 mL assay buffer and 20 μL 1M DTT. Add sufficient cathepsin S togive 2 nM final concentration.

Using the a distributor such as a Multidrop 384, add 30 μL/well to allwells of the assay plate and read in fluorescent spectrophotometer suchas an Ascent.

Fluorescent readings, (excitation and emission wavelengths 390 nm and460 nm respectively, set using bandpass filters) reflecting the extentof enzyme cleavage of the fluorescent substrate, notwithstanding theinhibitor, are linear rate fitted for each well.

Fitted rates for all wells for each inhibitor are fitted to thecompetitive inhibition equation using SigmaPlot 2000 to determine V, Kmand Ki values.

Cathepsin L Ki

The procedure above with the following amendments is used for thedetermination of Ki for cathepsin L.

The enzyme is commercially available human cathepsin L (for exampleCalbiochem). The substrate is H-D-Val-Leu-Lys-AMC available from Bahcem.The assay buffer is 100 mM sodium acetate 1 mM EDTA, pH5.5) The DMSOstock (10 mM in 100% DMSO) is diluted to 10% in assay buffer. Enzyme isprepared at 5 nM concentration in assay buffer plus 1 mM dithiothreitoljust before use. 2 μL of 10 mM inhibitor made up in 100% DMSO isdispensed into row A. 10 μL of 50 μM substrate (= 1/200 dilution of 10mM stock in DMSO, diluted in assay buffer).

Inhibition Studies

Potential inhibitors are screened using the above assay with variableconcentrations of test compound. Reactions were initiated by addition ofenzyme to buffered solutions of substrate and inhibitor. K_(i) valueswere calculated according to equation 1.

$\begin{matrix}{v_{0} = \frac{VS}{{K_{M}\left( {1 + \frac{I}{K_{i}}} \right)} + S}} & (1)\end{matrix}$

where v₀ is the velocity of the reaction, V is the maximal velocity, Sis the concentration of substrate with Michaelis constant of K_(M), andI is the concentration of inhibitor. The inhibition of cathepsin S,cathepsin K and cathepsin L exhibited by a selection of the compounds ofthe invention represented as Ki values is presented in Table 1.

TABLE 1 Example Ki Cat. S Ki Cat. K Ki Cat. L C1 12 770 11000 C2 8.3 5109700 C3 1.3 440 1700 C4 1.1 720 1700 C5 2.8 1100 5300 C6 2.4 960 3700 C76.4 2500 21000 C9 4.6 2100 520 C11 3.7 1200 2600 C12 2.7 1900 3200 C142.1 640 2800 A1 0.084 500 230 A2 0.3 630 1800 A3 0.1 140 180 A4 4.9 230012000 A7 0.88 650 260 A13 19 220 4300 A17 0.67 490 490 A24 1.2 300 3000A25 3.6 1300 14000 A28 31 15000 23000 A31 2.3 990 11000 A34 0.77 180 710A35 9.7 7500 18000 A38 7.6 4600 >200000 A43-1 1.3 280 4200

The compounds of formula I are thus potent inhibitors of cathepsin S andyet selective over the closely related cathepsin K and L.

Permeability

This experiment measures transport of inhibitors through the cells ofthe human gastroenteric canal. The assay uses the well known Caco-2cells with a passage number between 40 and 60.

Apical to Basolateral Transport

Generally every compound will be tested in 2-4 wells. The basolateraland the apical wells will contain 1.5 mL and 0.4 mL transport buffer(TB), respectively, and the standard concentration of the testedsubstances is 10 μM. Furthermore all test solutions and buffers willcontain 1% DMSO. Prior to the experiment the transport plates arepre-coated with culture medium containing 10% serum for 30 minutes toavoid nonspecific binding to plastic material. After 21 to 28 days inculture on filter supports, the cells are ready for permeabilityexperiments.

Transport plate no 1 comprises 3 rows of 4 wells each. Row 1 is denotedWash, row 2 “30 minutes” and row 3 “60 minutes”. Transport plate no 2comprises 3 rows of 4 wells, one denoted row 4 “90 minutes”, row 5 “120minutes and the remaining row unassigned.

The culture medium from the apical wells is removed and the inserts aretransferred to a wash row (No. 1) in a transport plate (plate no. 1) outof 2 plates without inserts, which have already been prepared with 1.5mL transport buffer (HBSS, 25 mM HEPES, pH 7.4) in rows 1 to 5. In A→Bscreening the TB in basolateral well also contains 1% Bovine SerumAlbumin.

0.5 mL transport buffer (HBSS, 25 mM MES, pH 6.5) is added to theinserts and the cell monolayers equilibrated in the transport buffersystem for 30 minutes at 37° C. in a polymix shaker. After beingequilibrated to the buffer system the Transepithelial electricalresistance value (TEER) is measured in each well by an EVOM chop stickinstrument. The TEER values are usually between 400 to 1000Ω per well(depends on passage number used).

The transport buffer (TB, pH 6.5) is removed from the apical side andthe insert is transferred to the 30 minutes row (No. 2) and fresh 425 μLTB (pH 6.5), including the test substance is added to the apical (donor)well. The plates are incubated in a polymix shaker at 37° C. with a lowshaking velocity of approximately 150 to 300 rpm.

After 30 minutes incubation in row 2, the inserts are moved to newpre-warmed basolateral (receiver) wells every 30 minutes; row 3 (60minutes), 4 (90 minutes) and 5 (120 minutes).

25 μL samples are taken from the apical solution after ˜2 minutes and atthe end of the experiment. These samples represent donor samples fromthe start and the end of the experiment.

300 μL will be taken from the basolateral (receiver) wells at eachscheduled time point and the post value of TEER is measured at the endthe experiment. To all collected samples acetonitrile will be added to afinal concentration of 50% in the samples. The collected samples will bestored at −20° C. until analysis by HPLC or LC-MS.

Basolateral to Apical Transport

Generally every compound will be tested in 2-4 wells. The basolateraland the apical wells will contain 1.55 mL and 0.4 mL TB, respectively,and the standard concentration of the tested substances is 10 μM.Furthermore all test solutions and buffers will contain 1% DMSO. Priorto the experiment the transport plates are precoated with culture mediumcontaining 10% serum for 30 minutes to avoid nonspecific binding toplastic material.

After 21 to 28 days in culture on filter supports the cells are readyfor permeability experiments. The culture medium from the apical wellsare removed and the inserts are transferred to a wash row (No. 1) in anew plate without inserts (Transport plate).

The transport plate comprises 3 rows of 4 wells. Row 1 is denoted “wash”and row 3 is the “experimental row”. The transport plate has previouslybeen prepared with 1.5 mL TB (pH 7.4) in wash row No. 1 and with 1.55 mLTB (pH 7.4), including the test substance, in experimental row No. 3(donor side).

0.5 mL transport buffer (HBSS, 25 mM MES, pH 6.5) is added to theinserts in row No. 1 and the cell monolayers are equilibrated in thetransport buffer system for 30 minutes, 37° C. in a polymix shaker.After being equilibrated to the buffer system the TEER value is measuredin each well by an EVOM chop stick instrument.

The transport buffer (TB, pH 6.5) is removed from the apical side andthe insert is transferred to row 3 and 400 μL fresh TB, pH 6.5 is addedto the inserts. After 30 minutes 250 μL is withdrawn from the apical(receiver) well and replaced by fresh transport buffer. Thereafter 250μL samples will be withdrawn and replaced by fresh transport bufferevery 30 minutes until the end of the experiment at 120 minutes, andfinally a post value of TEER is measured at the end of the experiment. A25 μL samples will be taken from the basolateral (donor) compartmentafter ˜2 minutes and at the end of the experiment. These samplesrepresent donor samples from the start and the end of the experiment.

To all collected samples acetonitrile will be added to a finalconcentration of 50% in the samples. The collected samples will bestored at −20° C. until analysis by HPLC or LC-MS.

Calculation

Determination of the cumulative fraction absorbed, FA_(cum), versustime. FA_(cum) is calculated from:

${FA}_{cum} = \Sigma_{\frac{C_{RI}}{C_{DI}}}$

Where C_(Ri) is the receiver concentration at the end of the interval iand C_(Di) is the donor concentration at the beginning of interval i. Alinear relationship should be obtained.

The determination of permeability coefficients (P_(app), cm/s) arecalculated from:

$P_{app} = \frac{\left( {k \cdot V_{R}} \right)}{\left( {A \cdot 60} \right)}$

where k is the transport rate (min⁻¹) defined as the slope obtained bylinear regression of cumulative fraction absorbed (FA_(cum)) as afunction of time (min), V_(R) is the volume in the receiver chamber(mL), and A is the area of the filter (cm²).

Reference Compounds

Category of absorption in man Markers absorption in man (%) PASSIVETRANSPORT Low (0-20%) Mannitol 16 Methotrexate 20 Moderate (21-75%)Acyclovir 30 High (76-100%) Propranolol 90 Caffeine 100 ACTIVE TRANSPORTAmino acid transporter L-Phenylalanine 100 ACTIVE EFFLUX PGP-MDR1Digoxin 30

Greater permeability through the gastrointestinal tissue is advantageousin that it allows for the use of a smaller dose to achieve similarlevels of exposure to a less permeable compound administered in a higherdose. A low dose is advantageous in that it minimizes the cost of goodsfor a daily dose, which is a crucial parameter in a drug which is takenfor protracted time periods.

All references referred to in this application, including patent andpatent applications, are incorporated herein by reference to the fullestextent possible.

Throughout the specification and the claims which follow, unless thecontext requires otherwise, the word ‘comprise’, and variations such as‘comprises’ and ‘comprising’, will be understood to imply the inclusionof a stated integer, step, group of integers or group of steps but notto the exclusion of any other integer, step, group of integers or groupof steps.

The application of which this description and claims forms part may beused as a basis for priority in respect of any subsequent application.The claims of such subsequent application may be directed to any featureor combination of features described herein. They may take the form ofproduct, composition, process, or use claims and may include, by way ofexample and without limitation, the following claims:

1. A compound of the formula IV:

wherein Pg is an amino protecting group; n is 2; and R^(2a) and R^(2b)are independently H or F.
 2. A compound of the formula 2a:

wherein Pg is an amino protecting group; n is 2; R^(2a) and R^(2b) areindependently H or F; and R³ is 1-methylcyclopentylmethyl or1-fluorocyclopentylmethyl.
 3. A compound of the formula 2b:

wherein n is 2; R^(2a) and R^(2b) are independently H or F; R³ is1-methylcyclopentylmethyl or 1-fluorocyclopentylmethyl; and R⁴ isC₁-C₆alkyl C₁-C₆haloalkyl or C₃-C₆cycloalkyl, wherein C₃-C₆cycloalkyl isoptionally substituted with methyl, CF₃ or one or two fluoro.
 4. Acompound according to any one of claims 1 to 3, wherein Pg is Boc, CBzor Fmoc.
 5. A compound according to any one of claims 1 to 3, whereinR^(2a) and R^(2b) are both H.
 6. A compound having the formula IV-a:


7. A process for the preparation of a compound of formula III,

wherein R^(2a) and R^(2b) are independently H or F; R^(3′) is CH₃ or F;R⁴ is C₁-C₆alkyl, C₁-C₆haloalkyl or C₃-C₆cycloalkyl, whereinC₃-C₆cycloalkyl is optionally substituted with methyl, CF₃ or one or twofluoro, comprising the step of oxidising the compound of formula 2b,

wherein n is 2; and R³ is 1-methylcyclopentylmethyl or1-fluorocyclopentylmethyl.
 8. The process according to claim 7, whereinthe compound of formula 2b is prepared by a process comprising the stepof removing the N-protecting group from a compound of formula 2a,followed by coupling of a P3 building block as outlined in the scheme:

wherein Pg is an amino protecting group and n, R^(2a), R^(2b), R³ and R⁴are as defined in claim
 7. 9. The process according to claim 8, whereinthe compound of formula 2a is prepared by a process comprising the stepof removing the N-protecting group from a compound of formula IV,followed by coupling of a P2 building block as outlined in the scheme:

wherein Pg, n, R^(2a), R^(2b) and R³ are as defined in claim
 8. 10. Theprocess according to any one of claims 7 to 9, for the preparation of acompound of formula IIIc:

wherein R^(2a) and R^(2b) are H, R³ is 1-fluorocyclopentylmethyl and R⁴is 1,1,1-trifluoro-2-methylpropan-2-yl.
 11. The process according to anyone of claims 7 to 9, for the preparation of a compound of formula IIId:

wherein R^(2a) and R^(2b) are H, R³ is 1-fluorocyclopentylmethyl and R⁴is 1,1-difluoroethyl.
 12. A process for the preparation of a compound ofclaim 3, comprising the step of removing the N-protecting group from thecompound of formula 2a, followed by coupling of a P3 building block asoutlined in the scheme:

wherein Pg is an amino protecting group; n is 2; R^(2a) and R^(2b) areindependently H or F; R³ is 1-methylcyclopentylmethyl or1-fluorocyclopentylmethyl; R⁴ is C₁-C₆alkyl, C₁-C₆haloalkyl orC₃-C₆cycloalkyl, wherein C₃-C₆cycloalkyl is optionally substituted withmethyl, CF₃ or one or two fluoro.
 13. The process according to claim 12,wherein the compound of formula 2a is prepared by a process comprisingthe step of removing of the N-protecting group from the compound offormula IV, followed by coupling of a P2 building block as outlined inthe scheme:

wherein Pg, n, R^(2a), R^(2b) and R³ are as defined in claim
 12. 14. Aprocess for the preparation of a compound of claim 2 comprising the stepof removal of the N-protecting group from the compound of formula IV,followed by coupling of a P2 building block as outlined in the scheme:

wherein Pg is an amino protecting group; n is 2; R^(2a) and R^(2b) areindependently H or F; and R³ is 1-methylcyclopentylmethyl or1-fluorocyclopentylmethyl.